EP4662318A1 - Pikfyve antisense oligonucleotides - Google Patents

Pikfyve antisense oligonucleotides

Info

Publication number
EP4662318A1
EP4662318A1 EP24713778.9A EP24713778A EP4662318A1 EP 4662318 A1 EP4662318 A1 EP 4662318A1 EP 24713778 A EP24713778 A EP 24713778A EP 4662318 A1 EP4662318 A1 EP 4662318A1
Authority
EP
European Patent Office
Prior art keywords
antisense oligonucleotide
modified
linkages
seq
nos
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24713778.9A
Other languages
German (de)
French (fr)
Inventor
Wen-Hsuan Chang
Justin K. ICHIDA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acurastem Inc
Original Assignee
Acurastem Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Acurastem Inc filed Critical Acurastem Inc
Publication of EP4662318A1 publication Critical patent/EP4662318A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0115—1-Phosphatidylinositol-3-phosphate 5-kinase (2.7.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3525—MOE, methoxyethoxy

Definitions

  • the present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods for treating, inhibiting, suppressing, and preventing neurological or neurodegenerative diseases with them.
  • ASOs PIKFYVE antisense oligonucleotides
  • the present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and their use in the treatment of neurodegenerative disorders.
  • ASOs PIKFYVE antisense oligonucleotides
  • the disclosure provides a single stranded ASO that suppresses the expression of PIKFYVE, wherein the ASO has a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the disclosure provides a single stranded ASO that suppresses the expression of PIKFYVE, wherein the ASO has a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725,
  • the nucleobase sequence of the ASO can comprise up to 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the nucleobase sequence of the ASO can comprise up to 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754,
  • the ASO can also be any of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376- 420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646- 2321.
  • the ASO can also be any of SEQ ID NOs: 1-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-17
  • the disclosure provides an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the disclosure provides an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698,
  • the oligonucleotide can comprise up to 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the oligonucleotide can comprise up to 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756
  • At least one internucleoside linkage is a modified intemucleoside linkage
  • the modified internucleoside linkage may be a phosphorothioate internucleoside linkage or a phosphodiester intemucleoside linkage.
  • At least one of the nucleosides may also be a modified nucleobase.
  • At least one nucleoside of the ASO may be a modified sugar moiety, where that modified sugar moiety can be a bicyclic sugar moiety, or the modified sugar moiety may comprise a 2'-O-methoxyethyl group.
  • the bicyclic sugar moiety comprises a 4'-CH(R)-O-2' bridge where the R group is, independently, H, C1-12 alkyl, or a protecting group.
  • the ASO is a gapmer (e.g., a MOE gapmer), where a gap segment may consist of 8 to 12 linked deoxynucleosides, a 5' wing segment consisting of 3 to 5 linked nucleosides, and a 3' wing segment consisting of 3 to 5 linked nucleosides.
  • the gap segment may be positioned between the 5' wing segment and the 3' wing segment, where a nucleoside of each wing segment comprises a modified sugar moiety (e.g., one with a 2'-O- methoxy ethyl group).
  • the oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 17
  • the disclosure provides a pharmaceutical composition comprising a PIKFYVE ASO of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
  • the pharmaceutical composition is suitable for parenteral administration, such as intracerebroventricular injection or intrathecal administration.
  • the disclosure provides a method of inhibiting, suppressing, or preventing expression of PIKFYVE in a patient (such as one having a neurological or neurodegenerative disease) by administering (for example by, intracerebroventricular injection or intrathecal administration) a PIKFYVE ASO or a pharmaceutical composition described herein (for instance, an effective amount thereof) to the patient.
  • the disclosure provides a method of treating a subject having a neurological or neurodegenerative disease by administering a therapeutically effective amount of a PIKFYVE ASO or a pharmaceutical composition described herein.
  • the disease is amyotrophic lateral sclerosis (ALS) (e.g., C9orf72-associated ALS).
  • ALS amyotrophic lateral sclerosis
  • the disease is frontotemporal dementia (FTD), such as FTD with TDP-43 pathology or FTD with tau pathology.
  • FTD frontotemporal dementia
  • the disease is C9orf72-associated FTD (C9-FTD).
  • the disease is microtubule-associated protein tau (MAPT)-associated FTD (MAPT- FTD), such as FTD with the V337M MAPT mutation.
  • MTT microtubule-associated protein tau
  • the disclosure provides a method of treating a subject having a PIKFYVE disease or disorder by administering a therapeutically effective amount of a PIKFYVE ASO or a pharmaceutical composition described herein.
  • FIGs. 1 A and IB predict skipping of exon 3 of PIKFYVE will generate a stop codon to cause early-termination of the translation of the PIKFYVE protein. represents a stop codon.
  • the sequence shown is taken from Ensembl Database (ENST00000264380).
  • FIGs. 2 A and 2B predict skipping of exon 5 of PIKFYVE will make major structural change to the PIKFYVE protein. represents a stop codon.
  • the sequence shown is taken from Ensembl Database (ENST00000264380).
  • unmodified versions of an ASO/sequence means an ASO/sequence that comprises only DNA or RNA nucleosides connected by phosphodiester linkages.
  • 2'-deoxynucleoside means a nucleoside comprising a 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA) and a nucleobase.
  • a 2'-deoxynucleoside may comprise a modified nucleobase and a furanosyl sugar moiety or may comprise an RNA nucleobase (uracil) and a furanosyl sugar moiety.
  • 2'-substituted nucleoside means a nucleoside comprising a 2'- substituted sugar moiety.
  • 2'-substituted in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.
  • antisense molecule means an oligomeric nucleic acid or oligomeric duplex capable of achieving at least one antisense activity.
  • the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity).
  • the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints.
  • the expression “from about 2 to about 4” also discloses the range “from 2 to 4.”
  • the term “about” may refer to plus or minus 10% of the indicated number.
  • “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1.
  • Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • bicyclic sugar or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure.
  • the first ring of the bicyclic sugar moiety is a furanosyl moiety.
  • the bicyclic sugar moiety does not comprise a furanosyl moiety.
  • bicyclic nucleoside or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • chirally enriched population means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers.
  • the molecules are modified oligonucleotides. In some aspects, the molecules are compounds comprising modified oligonucleotides.
  • complementary in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions.
  • Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another.
  • Complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); cytosine (C) and guanine (G); and 5-methylcytosine (mC) and guanine (G).
  • Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • “fully complementary” or “100% complementary” in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
  • gapmer means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
  • the internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”
  • wings refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2'-deoxyfuranosyl.
  • MOE gapmer indicates a gapmer having a sugar motif of 2'- MOE nucleosides in both wings and a gap of 2'-deoxynucleosides. Unless otherwise indicated, a MOE gapmer may comprise one or more modified internucleoside linkages and/or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications. Table 2 and Table 6, below, provides exemplary MOE-gapmers.
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.
  • modified oligonucleotides comprise or consist of a region having a gapmer motif, which is defined by two external regions or “wings” and a central or internal region or “gap.”
  • the three regions of a gapmer motif include the “5’ wing”, the “gap” and the “3’ wing” which form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction).
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar motifs of the two wings are the same as one another (symmetric gapmer).
  • the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).
  • the wings of a gapmer comprise 1-5 nucleosides.
  • each nucleoside of each wing of a gapmer is a modified nucleoside.
  • the gap of a gapmer comprises 7-12 nucleosides (e.g., 10 nucleosides). In some aspects, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxy nucleoside.
  • the gapmer is a deoxy gapmer.
  • the nucleosides on the gap side of each wing/gap junction are unmodified 2'-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides.
  • each nucleoside of the gap is an unmodified 2'-deoxy nucleoside.
  • each nucleoside of each wing of a gapmer is a modified nucleoside.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif.
  • each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety.
  • each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif.
  • a fully modified oligonucleotide is a uniformly modified oligonucleotide.
  • each nucleoside of a uniformly modified oligonucleotide comprises the same 2'-modification.
  • “Inhibit” as used herein refers to the ability to substantially antagonize, prohibit, prevent, suppress, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of the activity of a particular agent (e.g., infectious agent) or disease.
  • a particular agent e.g., infectious agent
  • internucleoside linkage is the covalent linkage between adjacent nucleosides in an oligonucleotide.
  • modified internucleoside linkage means any intemucleoside linkage other than a phosphodiester internucleoside linkage.
  • Phosphorothioate linkage is a modified internucleoside linkage in which one of the nonbridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom.
  • nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage.
  • the two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative phosphorus-containing intemucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates or other alkylphosphonates, phosphoramidates, and phosphorothioates, and phosphorodithioates.
  • Modified intemucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • intemucleoside linkages having a chiral center include, but are not limited to, alkylphosphonates and phosphorothioates.
  • Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereo-random intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations.
  • populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereo-random.
  • modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration.
  • populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleoside linkages in a particular, independently selected stereochemical configuration.
  • the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population.
  • the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population.
  • Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); Chapter 10 of Locked Nucleic Acid Aptamers in Nucleic Acid and Peptide Aptamers: Methods and Protocols v 535, 2009 by Barciszewski et al., editor Gunter Mayerand; and WO 2017/015555.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In some aspects, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Rp) configuration.
  • MOE means methoxy ethyl.
  • 2'-M0E means a -OCH2CH2OCH3 group at the 2' position of a furanosyl ring.
  • a “neurological disease” is any disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons.
  • a neurological disease may be a neurodegenerative disease.
  • the neurodegenerative disease may result in motor neuron degeneration, for example.
  • the neurological disease may be amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, or frontotemporal dementia, for example.
  • Further examples of neurological diseases include, but are not limited to, Parkinson’s disease, multiple sclerosis, peripheral myopathy, Rasmussen’s encephalitis, attention deficit hyperactivity disorder, autism, central pain syndromes, anxiety, and/or depression, for example.
  • the neurological disease may be associated with aberrant endosomal trafficking.
  • endosomal pathways and endosomes are necessary components for the recycling or breakdown of membrane-bound proteins, trafficking of Golgi-associated proteins, and the extracellular release of proteins in exosomes. These processes aid neurotransmission and drive a balance between recycling and degradation of synaptic vesicles or neurotransmitter receptors, for example.
  • the neurological disease may be associated with aberrant lysosome degradation. Alterations in the lysosome degradation may be present in the neurological disease, such as a neurodegenerative disease. Cathepsin imbalance during aging and age-related diseases may provoke deleterious effects on central nervous system (CNS) neurons and lysosomes may be sites for the unfolding and partial degradation of membrane proteins or their precursors that subsequently become expelled from a cell, or are released from dead cells and accumulate as pathological entities.
  • CNS central nervous system
  • a health care professional may diagnose a subject as having a disease associated with motor neuron degeneration by the assessment of one or more symptoms of motor neuron degeneration.
  • a physical exam may be followed by a thorough neurological exam.
  • the neurological exam may assess motor and sensory skills, nerve function, hearing and speech, vision, coordination and balance, mental status, and changes in mood or behavior.
  • Non-limiting symptoms of a disease associated with a neurological disease may be weakness in the arms, legs, feet, or ankles; slurring of speech; difficulty lifting the front part of the foot and toes; hand weakness or clumsiness; muscle paralysis; rigid muscles; involuntary jerking or writing movements (chorea); involuntary, sustained contracture of muscles (dystonia); bradykinesia; loss of automatic movements; impaired posture and balance; lack of flexibility; tingling parts in the body; electric shock sensations that occur with movement of the head; twitching in arm, shoulders, and tongue; difficulty swallowing; difficulty breathing; difficulty chewing; partial or complete loss of vision; double vision; slow or abnormal eye movements; tremor; unsteady gait; fatigue; loss of memory; dizziness; difficulty thinking or concentrating; difficulty reading or writing; misinterpretation of spatial relationships; disorientation; depression; anxiety; difficulty making decisions and judgments; loss of impulse control; difficulty in planning and performing familiar tasks; aggressiveness; irritability; social
  • Tests may be performed to rule diseases and disorders that may have symptoms similar to those of neurological diseases, measure muscle involvement, assess neuron degeneration.
  • tests are electromyography (EMG); nerve conduction velocity study; laboratory tests of blood, urine, or other substances; magnetic resonance imaging (MRI); magnetic resonance spectroscopy; muscle or nerve biopsy; transcranial magnetic stimulation; genetic screening; x-rays; fluoroscopy; angiography; computed tomography (CT); positron emission tomography; cerebrospinal fluid analysis; intrathecal contrast-enhanced CT scan; electroencephalography; electronystagmography; evoked response; polysomnogram; thermography; and ultrasound.
  • EMG electromyography
  • MRI magnetic resonance imaging
  • CT computed tomography
  • CT positron emission tomography
  • cerebrospinal fluid analysis intrathecal contrast-enhanced CT scan
  • electroencephalography electronystagmography
  • evoked response polysomnogram
  • thermography thermography
  • a health care professional may also assess the patient’s family history of diseases associated with motor neuron degeneration and make a diagnosis in part based on a familial history of neurological diseases.
  • a healthcare professional may diagnose a disease associated with neurological disease in a subject after the presentation of one or more symptoms.
  • Neurodegenerative diseases result in the progressive destruction of neurons that affects neuronal signaling.
  • a neurodegeneration may be amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, Friedreich’s ataxia, Lewy body disease, Parkinson’s disease, spinal muscle atrophy, primary lateral sclerosis, progressive muscle atrophy, progressive bulbar palsy, and pseudobulbar palsy.
  • Diseases associated with motor neuron degeneration may be a condition that results in the progressive destruction of motor neurons that interferes with neuronal signaling to the muscles, leading to muscle weakness and wasting.
  • upper motor neurons transmit signals from the brain to lower motor neurons in the brain stem and spinal cord, which then transmit the signal to the muscles to result in voluntary muscle activity.
  • the destruction of upper and lower motor neurons affects activity such as breathing, talking, swallowing, and walking, and overtime these functions can be lost.
  • motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscle atrophy, progressive bulbar palsy, and pseudobulbar palsy.
  • Neuronal hyperexcitability may occur when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are over-activated by excess glutamate or by other compounds or neurotransmitters acting on the glutamate receptors.
  • Excitotoxicity may result from neuronal hyperexcitability.
  • Exci totoxi city is the pathological process by which nerve cells are damaged or killed by excessive stimulation. The excessive stimulation allows high levels of calcium ions (Ca 2+ ) to enter the cell. Ca 2+ influx into cells activates a number of enzymes, including phospholipases, endonucleases, and proteases such as calpain. These enzymes can damage cell structures such as components of the cytoskeleton, membrane, and DNA.
  • Neuronal hyperexcitability may be involved in spinal cord injury, stroke, traumatic brain injury, hearing loss (through noise overexposure or ototoxicity), epilepsy, painful neuropathies, attention deficit hyperactivity disorder, autism, central pain syndromes, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, schizophrenia, Rasmussen’s encephalitis, Huntington’s disease, alcoholism or alcohol withdrawal and especially over-rapid benzodiazepine withdrawal, and also Huntington's disease.
  • Other common conditions that cause excessive glutamate concentrations around neurons are hypoglycemia. Blood sugars are the primary glutamate removal method from inter-synaptic spaces at the NMDA and AMPA receptor site.
  • non-bicyclic modified sugar moiety means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
  • nucleobase means an unmodified nucleobase or a modified nucleobase.
  • an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G).
  • a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase or modified nucleobase.
  • a “5 -methylcytosine” or “mC” is a modified nucleobase.
  • a universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.
  • nucleobase sequence means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.
  • modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase.
  • modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase.
  • modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines.
  • modified nucleobases are selected from: 2- aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (- OC-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8- substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5- halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoaden
  • nucleobases include tricyclic pyrimidines, such as 1,3- diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3- diazaphenoxazine-2-one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in U.S. Patent No.
  • nucleoside means a compound comprising a nucleobase and a sugar moiety.
  • the nucleobase and sugar moiety are each, independently, unmodified or modified.
  • modified nucleoside means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.
  • Modified nucleosides include abasic nucleosides, which lack a nucleobase.
  • Linked nucleosides are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are presented between those that are linked).
  • oligomeric compound means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
  • An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired.
  • a “singled stranded oligomeric compound” is an unpaired oligomeric compound.
  • oligomeric duplex means a duplex formed by two oligomeric compounds having complementary nucleobase sequences.
  • oligonucleotide means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified.
  • the internucleoside linkages may be any described herein. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides.
  • modified oligonucleotide means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified.
  • unmodified oligonucleotide means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.
  • PIKFYVE also known in the art as “phosphatidylinositol-3-phosphate 5-kinase type III” or “PIPKIII”, is a FYVE finger-containing phosphoinositide kinase encoded by the PIKFYVE gene.
  • PIKFYVE is a highly evolutionarily conserved lipid kinase and also has protein kinase activity, which regulates endomembrane homeostasis and plays a role in the biogenesis of endosome carrier vesicles from early endosomes.
  • PIKFYVE-mediated conversion of PI3P to PI(3,5)P2 blocks recruitment of the protein EEA1. The recruitment is blocked, because PIP3 is needed to form a platform with RAB5 that enables anchoring of EEA1 to early endosomes. EEA1 then drives fusion with endocytic and other endosomal vesicles.
  • a “PIKFYVE disease or disorder” includes lysosomal degradation diseases and disorders mediated by PIKFYVE.
  • the a PIKFYVE disease or disorder includes, but is not limited to, amyloid diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis and chronic hemodialysis-related amyloid), multiple sclerosis, and an MPS disorder (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX).
  • amyloid diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis and chronic hemodialysis-related amyloid
  • MPS disorder such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA,
  • the diseases are autoimmune disorders (such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, Ankylosing spondylitis, psoriasis, psoriatic arthritis, adult still disease, Becet syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease), or CNS disorders (such as spongiform encephalopathies (Creutzfeld- Jakob, Kuru, Mad Cow)).
  • autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, Ankylosing spondylitis, psoriasis, psoriatic arthritis, adult still disease, Becet syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease
  • CNS disorders such as spongiform encephalopathie
  • compositions and methods of the disclosure can be used to treat individuals with lysosomal storage diseases comprising administering to a subject in need of treatment a therapeutically effective amount of a PIKfyve ASO or pharmaceutical composition described herein.
  • the ASOs and compositions of the disclosure decrease or inhibit the activity of PIKfyve and alters the biogenesis, function or dynamics of the endosomal or lysosomal systems in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases.
  • the ASOs and compositions target, decrease or inhibit the activity of PIKfyve thus altering the biogenesis, functions, or dynamics of the endoplasmic reticulum or Golgi apparatus in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases.
  • the disease is a neurological disorder.
  • sugar moiety means an unmodified sugar moiety or a modified sugar moiety.
  • the superscript prime symbol (') is used to describe the numbering of a sugar in a nucleoside or nucleotide (the nucleobase positions are numbered without the prime). When describing the sugar only, the prime symbol is not used.
  • unmodified sugar moiety means a 2-0H(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).
  • Unmodified sugar moi eties have one hydrogen at each of the 1, 3, and 4 positions, an oxygen at the 3 position, and two hydrogens at the 5 position.
  • modified sugar moiety or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.
  • modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety.
  • a modified furanosyl sugar moiety is a 2-substituted sugar moiety.
  • Such modified furanosyl sugar moieties include bicyclic sugars and nonbicyclic sugars.
  • modified sugar moieties are nonbicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure.
  • Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2, 4, and/or 5 positions.
  • one or more non-bridging substituent of nonbicyclic modified sugar moieties is branched.
  • 2-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2-F, 2-OCHs (“OMe” or “O-methyl”), and 2- O(CH 2 ) 2 OCH 3 (“MOE”).
  • non-bicyclic modified sugar moieties examples include but are not limited to alkoxy (e.g., methoxy), and alkyl.
  • alkoxy e.g., methoxy
  • 5-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5-methyl (R or S), 5-vinyl, and 5- methoxy.
  • non-bicyclic modified sugar moieties comprise more than one nonbridging sugar substituent, for example, 2-F-5-methyl sugar moieties and the like.
  • a nonbridging 2'-substituent group selected from: F, NH 2 , N3, OCF3, OCH
  • a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a nonbridging 2'-substituent group selected from: F, OCH3, and OCH 2 CH 2 OCH 3 .
  • modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4 and the 2 furanose ring atoms.
  • a mammal e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse
  • a non-human primate for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.
  • the subject may be a human or a non-human.
  • the subject or patient is a human.
  • the patient has a neurological disease due to a mutation in the C9ORF72 gene (for instance, the patient may be haploinsufficient for the C9ORF72 gene (e.g., one which results in a 50% or greater reduction in C9ORF72 protein activity), or the C9ORF72 gene may comprises a GGGGCC repeat expansion (such as a (GGGGCC)n (SEQ ID NO: 1565) hexanucleotide expansion in C90RF72)).
  • the variable “n” can be at least 30.
  • a “therapeutically effective amount,” or “effective dosage” or “effective amount” as used interchangeably herein unless otherwise defined, means a dosage of a drug effective for periods of time necessary, to achieve the desired therapeutic result.
  • An effective dosage may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, mammal, or human, such as reducing and/or inhibiting the function of a receptor.
  • a therapeutically effective amount may be administered in one or more administrations (e.g., the agent may be given as a preventative treatment or therapeutically at any stage of disease progression, before or after symptoms, and the like), applications or dosages and is not intended to be limited to a particular formulation, combination or administration route. It is within the scope of the present disclosure that the drug may be administered at various times during the course of treatment of the subject. The times of administration and dosages used will depend on several factors, such as the goal of treatment (e.g., treating v. preventing), condition of the subject, etc. and can be readily determined by one skilled in the art.
  • treat refers to administering a composition or agent described herein to the subject, such that at least one symptom of a disease or disorder is healed, alleviated, relieved, altered, remedied, reduced, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, reduce, ameliorate, and/or improve one or more symptoms associated with a disease or disorder. The treatment may inhibit deterioration or worsening of a symptom associated with the disease or disorder.
  • the herein described methods of treatment may comprise administering to a subject in need thereof a composition comprising an effective amount of one or more antisense oligonucleotides that treats neurological diseases by inhibiting or suppressing PIKFYVE expression.
  • the one or more antisense oligonucleotides may decrease or inhibit neurodegeneration.
  • the one or more antisense oligonucleotides may decrease neuronal hyperexcitability. Reducing PIKFYVE mRNA and PIKFYVE protein levels suppresses neurodegeneration driven by toxic TDP-43 aggregates, DPR aggregates (for example, in C9ORF72-ALS patients), and by promoting the retention of TDP-43 in the nucleus. Delivery of the ASOs targeting PIKFYVE mRNA described herein reduces PIKFYVE protein levels.
  • the composition may inhibit kinase activity by inhibiting expression of a kinase.
  • the composition may inhibit PIKFYVE kinase activity or expression.
  • the one or more antisense oligonucleotides can be combined with small molecule therapeutic agents (such as apilimod and/or YM201636).
  • the disclosure provides oligonucleotides (modified or unmodified) that can be used to modulate PIKFYVE expression.
  • Table 1 provides (5' to 3') generic sequence of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure.
  • Table 6 provides (5' to 3') modified sequence of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure.
  • Unmodified sequences of Table 6 are additionally envisioned as generic sequences of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure.
  • the ASO sequences of SEQ ID NOs: 1-49 are predicted to skip exon 3 of PIKFYVE efficiently, and generate a stop codon to cause early- termination of PIKFYVE translation , as shown in FIGs. 1 A and IB.
  • the ASO sequences of SEQ ID NOs: 50-80 are predicted to skip exon 5 of PIKFYVE efficiently, and make a major structural change to the protein rendering it non-functional, as shown in FIGs. 2A and 2B.
  • the ASO sequences SEQ ID NOs: 81-328, 330-373, 376-420, 423-479 have low acute neurotoxicity scores as determined by the method described in Hagedorn et al., Nucleic Acid Therapeutics, 2022, 32(3): 151-162 (DOI: 10.1089/nat/2021/0071).
  • the ASO sequences SEQ ID NOs: 307, 308, 330, 424, 480-540, 542-619, 621-685, 688-782, and the unmodified versions of SEQ ID NOs: 1646-2321 have low numbers of predicted off-target binding sites as determined by the GGGenome search tool using 2 mismatches/gaps.
  • the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 in Table 1; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 in Table 1 or and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-16
  • the modified oligonucleotide is at least 80% to 100% (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100%; or any numerical range or value between any of the foregoing values) identical to any of the sequences comprising or consisting of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321.
  • the modified oligonucleotide is at least 80% to 100% (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100%; or any numerical range or value between any of the foregoing values) identical to any of the sequences comprising or consisting of SEQ ID NOs: 1-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 17
  • sequences provided in Table 1 and the unmodified versions of the sequences of Table 6 can be used to design antisense molecules for inhibition of PIKFYVE expression.
  • gapmer oligonucleotides can be designed using the sequences in Table 1 and the unmodified versions of the sequences of Table 6, and can comprise a 5'-wing of about 3-5 nucleotides, a 3'-wing of about 3-5 nucleotides and a gap region comprising 8-12 consecutive deoxyribonucleosides of any one of the sequences of Table 1 and the unmodified versions of the sequences of Table 6.
  • an oligonucleotide of the disclosure comprises a gapmer having a gap segment of at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 in Table 1; and the unmodified versions of SEQ ID NOs: 1646-2321; flanked by a 5' and 3' wing segments, wherein the gap segment is located between the 5' and 3' wing segments and wherein each of the wing segments comprises a modified sugar.
  • the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length.
  • an oligonucleotide of the disclosure comprises a 5' wing segment comprising modified sugars and having the nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542- 619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321, followed by a 3' wing segment comprising modified sugars and having the nucleobase sequence of the last
  • an oligonucleotide of the disclosure comprises a gapmer having a gap segment of at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425- 498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 in Table 1 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702
  • an oligonucleotide of the disclosure comprises a 5' wing segment comprising modified sugars and having the nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 17
  • the 5' and/or 3' wings can comprise the following chemistries: 2'-OMe, 2'-M0E, LNA or DNA, by themselves or used in combination with one another.
  • the backbone linkage of the 5' and/or 3' wings can be phosphorothioate or a mixture of phosphodiester and phosphorothioate. Linkages in the gap region can be phosphorothioate.
  • the oligonucleotide is single stranded. In some aspects, the oligonucleotide comprises or is complexed with a moiety that neutralizes charge on the oligonucleotide to promote uptake and transfer across a cell membrane.
  • the ASO sequences of SEQ ID NOs: 1-80 in Table 1 have the following 5-8-5 motif: 2MOE*2MOE-2MOE-2MOE-2MOE-N*N*N*N*N*N*N*N*N*2MOE- 2MOE-2MOE*2MOE*2MOE where (i) 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E), (ii) N is a nucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage.
  • 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E)
  • N is a nucleobase
  • the asterisk (*) refers to a phosphorothi
  • Table 2 below shows this motif on SEQ ID NOs: 1-80 (here SEQ ID NOs: 783-862).
  • ASO sequences of SEQ ID NOs: 783-831 skip exon 3 of PIKFYVE efficiently.
  • ASO sequences of SEQ ID NOs: 832-862 skip exon 5 of PIKFYVE efficiently.
  • the ASO sequences of SEQ ID NOs: 1-782, for example 81-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782, in Table 1 and t the unmodified versions of SEQ ID NOs: 1646-2321 have the following 5-10-5 motif: 2MOE*2MOE-2MOE-2MOE- 2MOE-N*N*N*N*N*N*N*N*N*N*N*N*N*N*2MOE-2MOE-2MOE*2MOE*2MOE where (i) 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E), (ii) N is a nucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage.
  • SEQ ID NOs: 1-782 shows this motif on SEQ ID NOs: 1-782, for example 81-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 (here SEQ ID NOs: 783-1564, for example 863- 1110, 1112-1155, 1158-1202, 1205-1322, 1324-1401, 1403-1467, 1470-1564).
  • N N*N*N*N*N*N*N*N*N*N*N*N*N*N*N*2MOE-2MOE-2MOE*2MOE*2MOE
  • 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E)
  • N is a nucleobase
  • the asterisk (*) refers to a phosphorothioate linkage
  • the dash (-) refers to a phosphodiester linkage.
  • Table 2 The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Gapmer design: 5'- five 2'-methoxyethylribose nucleotides - ten DNA nucleotides - five 2'- methoxy ethylribose nucleotides - 3'; Capital letters are 2'-methoxyethylribose nucleosides; lower case are DNA nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
  • the ASO sequences of SEQ ID NOs: 1-49 in Table 1 have the following motif: 2MOE*2MOE-2MOE-2MOE-2MOE-2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE*2MOE*2MOE*2MOE*2MOE where (i) 2MOE is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) the asterisk (*) refers to a phosphorothioate linkage, and (iii) the dash (-) refers to a phosphodiester linkage.
  • Table 3 The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Design: 5'- eighteen 2'-methoxyethylribose nucleotides - 3'; Capital letters are 2'- methoxyethylribose nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
  • the ASO sequences of SEQ ID NOs: 50-80 in Table 1 have the following motif: 2MOE*2MOE-2MOE-2MOE-2MOE-2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE*2MOE*2MOE*2MOE*2MOE where (i) 2MOE is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) the asterisk (*) refers to a phosphorothioate linkage, and (iii) the dash (-) refers to a phosphodiester linkage.
  • 2MOE is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE)
  • the asterisk (*) refers to a phosphorothioate link
  • Table 4 below shows this motif on SEQ ID NOs: 50-80 (here SEQ ID NOs: 1615-1645).
  • ASO sequences of Table 4 skip exon 5 of PIKFYVE efficiently.
  • an ASO of a sequence set forth in SEQ ID NOs: 50-80 skip exon 5 of PIKFYVE efficiently.
  • Table 4 The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Design: 5'- eighteen 2'-methoxyethylribose nucleotides - 3'; Capital letters are 2'- methoxyethylribose nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
  • the PIKFYVE kinase antisense or inhibitory nucleic acids of the disclosure can inhibit the expression and thus the activity associate with PIKFYVE.
  • the PIKFYVE kinase antisense or inhibitory nucleic acids can include any combination of the oligonucleotides set forth in Tables 1, 2, 3, 4, 5, and 6 and sequences that are 98%-99% identical thereto.
  • PIKFYVE ASOs described herein such as SEQ ID NOs: 783-1564, for example 783-1110, 1112-1155, 1158-1202, 1205-1322, 1324-1401, 1403-1467, 1470-1564, and 1566- 2321, suppress PIKFYVE mRNA expression with minimal off-target binding.
  • the PIKFYVE ASOs described herein such as SEQ ID NOs: 783- 1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, and 1566-2321, for example SEQ ID NOs: 863-1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563; suppress PIKFYVE mRNA expression with minimal off-target binding.
  • Methods of treatment may include any number of modes of administering a disclosed composition.
  • Modes of administration may include aqueous, lipid, oily or other solutions, solutions in simulated cerebrospinal fluid, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions and the like.
  • an ASO of the disclosure will be administered directly to the CNS of the subject.
  • the formulation or composition will be sterile and more preferably be suitable for injection.
  • the following formulations and methods are merely exemplary and are in no way limiting.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations may be presented in unit-dose or multidose sealed containers, such as ampules and vials, and may be stored as liquids or in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulation may be provided in a pre-filled syringe.
  • Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed one or more antisense or inhibitory nucleic acids and compositions. Sequential administration includes administration before or after the disclosed one or more antisense or inhibitory nucleic acids or compositions. In some aspects, the additional therapeutic agent or agents may be administered in the same composition as the disclosed one or more antisense or inhibitory nucleic acids. In some aspects, there may be an interval of time between administration of the additional therapeutic agent and the disclosed one or more antisense or inhibitory nucleic acids. In some aspects, administration of an additional therapeutic agent with a disclosed one or more antisense or inhibitory nucleic acids may allow lower doses of the other therapeutic agents and/or administration at less frequent intervals.
  • the one or more antisense or inhibitory nucleic acids of the disclosure and the other active ingredients may be used in lower doses than when each is used singly.
  • the pharmaceutical compositions of the disclosure include those that contain one or more other active ingredients, in addition to one or more antisense or inhibitory nucleic acids of the disclosure.
  • the above combinations include combinations of one or more antisense or inhibitory nucleic acids of the disclosure not only with one other active compound, but also with two or more other active compounds.
  • the compound of the disclosure may be combined with a variety of drugs to treat neurological diseases.
  • the antisense oligonucleotide may be covalently linked to another oligonucleotide, such as one with a target other than PIKFYVE.
  • the antisense oligonucleotide may be covalently linked to an antibody.
  • the disclosed one or more antisense or inhibitory nucleic acids can be combined with the following, but are not limited, anticholinergic drugs, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, pain medications, and/or stimulants.
  • Additional types of therapy and treatment include, but are not limited to digital communication devices, feeding tubes, mechanical ventilation, nutritional support, deep brain stimulation, occupational therapy, physical therapy, and/or speech therapy.
  • composition(s) may be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which may be a human or non-human).
  • the pharmaceutical compositions may comprise a carrier (e.g., a pharmaceutically acceptable carrier). Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the composition of the present disclosure.
  • the pharmaceutical compositions may include a therapeutically effective amount or a prophylactically effective amount of the antisense oligonucleotide.
  • a therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of one or more antisense or inhibitory nucleic acids of the disclosure are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • compositions may include one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ring
  • compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal, intracerebroventricular, or intraventricular, administration.
  • the antisense or inhibitory nucleic acid is administered by intravenous, intraperitoneal, or as a bolus injection or administered directly into the target organ.
  • the antisense or inhibitory nucleic acid is administered intrathecally or intra-cerebroventricular as a bolus injection.
  • Carriers for systemic administration typically include at least one of solvent, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.
  • Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as com starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose.
  • the amount of binder(s) in a systemic composition is typically about 5 to about 50%.
  • Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins.
  • the amount of disintegrant(s) in a systemic composition is typically about 0.1 to about 10%.
  • Suitable colorants include a colorant such as an FD&C dye.
  • the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.
  • Suitable flavors include menthol, peppermint, and fruit flavors.
  • the amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.
  • Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E.
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluene
  • vitamin E The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.
  • Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate.
  • the amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.
  • Suitable glidants include silicon dioxide.
  • the amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.
  • Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions.
  • the amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.
  • Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate.
  • the amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.
  • Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware.
  • Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239.
  • the amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.
  • compositions and formulations for parenteral, intrathecal, intra-cerebroventricular, or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • an intrathecal cerebrospinal fluid (CSF) catheter can be used to deliver antisense formulations of the disclosure.
  • the catheter can be inserted at the L3 or L4 vertebrae. The distal tip of the catheter extends within the intrathecal space to approximately the LI vertebrae.
  • Antisense oligonucleotides are dissolved in saline, are sterilized by filtration, and are administered at 0.33 ml/min in a 1.0 ml volume followed by a 0.5 ml sterile water flush. Total infusion time is 4.5 min.
  • compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.
  • the amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the medicament.
  • Techniques and compositions for making dosage forms useful in the methods of this disclosure are described in the following references: Modem Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).
  • candidate antisense or inhibitory nucleic acids may be conducted by means known to one of ordinary skill in the art.
  • the candidate one or more antisense or inhibitory nucleic acids may be administered to a mammal, such as a mouse or a rabbit.
  • the mammal may be administered, by any route deemed appropriate, a dose of a candidate antisense or inhibitory nucleic acids.
  • Conventional methods and criteria can then be used to monitor animals for signs of reduction or improvement of motor neuron activity and/or expression or activity of PIKFYVE gene or protein, respectively.
  • the results obtained in the presence of the candidate antisense or inhibitory nucleic acids can be compared with results in control animals that are not treated with the candidate antisense or inhibitory nucleic acids.
  • Dosing studies may be performed in, or in conjunction with, the herein described methods for identifying one or more antisense or inhibitory nucleic acids capable of treating a neurological disease and/or any follow-on testing of candidate antisense or inhibitory nucleic acids in vivo.
  • One of skill in the art of medicine may determine the appropriate dosage of one or more antisense or inhibitory nucleic acids. The dosage may be determined by monitoring the subject for signs of disease inhibition or amelioration. The dosage may be increased or decreased to obtain the desired frequency of treatment.
  • the toxicity and efficacy of one or more antisense or inhibitory nucleic acids may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. determining the lethal dose to 50% of the population (LD50) and the dose therapeutically effective in 50% of the population (ED50).
  • the dose ratio of LD50/ED50 is the therapeutic index and, indicating the ratio between the toxic and therapeutic effects.
  • a delivery system may be designed to help prevent toxic side effects, by delivering the one or more antisense or inhibitory nucleic acids to specific targets, e.g., delivered specifically to motor or central nervous system neurons.
  • the optimal dose of the one or more antisense or inhibitory nucleic acids may be determined based on results of clinical electrophysiology or electromyography to analyze excitability in peripheral nerves, for example.
  • the dosage for use in humans may be determined by evaluating data obtained from animal studies and cell culture assays.
  • the preferred dosage will have little or no toxicity and include the ED50.
  • the dosage may vary depending on the dosage form and route of administration.
  • the dosage may be estimated initially in cell culture.
  • a dose may be formulated in animal models that includes the concentration of the test compound which achieves a half maximal inhibition of symptoms (LD50) as determined in cell culture. Such information obtained from cell cultures and animal models may be used to more accurately determine useful doses in humans.
  • Small molecule inhibitors of PIKFYVE kinase and antisense oligonucleotides (ASOs) that suppress PIKFYVE expression can prevent the degeneration of human and mouse neurons that carry a mutation in the C9ORF72 gene that leads to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • ASOs are an attractive therapeutic option for neurodegenerative diseases because of their ease of delivery to the central nervous system and their relatively low exposure to the periphery. These properties maximize target engagement in the central nervous system and minimize undesired target engagement or off-target effects in the periphery.
  • the disclosure provides novel antisense oligonucleotide (ASO) sequences targeting the PIKFYVE gene that can suppress PIKFYVE expression in human cells.
  • PIKFYVE ASOs can also rescue the survival of motor neurons derived from sporadic ALS patients.
  • PIKFYVE ASOs can lower levels of neurotoxic dipeptide repeat protein aggregates derived from the C9ORF72 repeat expansion in vivo in mice.
  • ASOs are designed (see Tables 2, 3, 4, and 6) and are synthesized as MOE gapmers, which contains sugar and linkage modifications that increase nuclease resistance and melting temperature while maintaining the ability to be used as a substrate of RNase H.
  • MOE gapmers contains sugar and linkage modifications that increase nuclease resistance and melting temperature while maintaining the ability to be used as a substrate of RNase H.
  • the ability of each ASO to suppress PIKFYVE RNA levels is tested by transfecting them into human embryonic kidney 293T cells with Lipofectamine 2000 at a concentration of 100 nM and measuring PIKFYVE expression 7 days after transfection.
  • a control NCASO
  • the relative PIKFYVE expression shown is an average of three technical replicates and values are calculated by normalizing to a GAPDH control.
  • VPIKFYVE BAC mice receive 25 pg of negative control ASO or test compound by intracerebroventricular (ICV) injection at Pl (postnatal day 1) and tissue samples are collected 14 days post-treatment.
  • ICV intracerebroventricular
  • ASOs of SEQ ID NOs. 863-1261 were determined to have low acute neurotoxicity scores. The results of the study are shown below in Table 5. TABLE 5
  • an ASO of a sequence set forth in SEQ ID NOs: 81-306, 310-328, 332- 373, 376-420, 423, and 425-479 comprise a low acute neurotoxicity score.
  • the ASO’s of Table 6 have the following Gapmer design: 5'- five 2'- methoxy ethylribose nucleotides - ten DNA nucleotides - five 2'-methoxy ethylribose nucleotides - 3'; wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, sixteenth, and seventeenth linkages are phosphodiester linkages (the same Gapmer design as Table 2). Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

The present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods for treating, inhibiting, suppressing, and preventing neurological diseases with them.

Description

PIKF YVE ANTISENSE OLIGONUCLEOTIDES
REFERENCE TO RELATED APPLICATIONS
[0001] This Application claims the priority benefit of U.S. Provisional Application No. 63/483,517, filed on February 6, 2023. The entire disclosure of the above-referenced application is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence listing (Name: 3817_225PC01_SequenceListing_ST26.xml; Size: 5,488,433 bytes; and Date of Creation: February 6, 2024) is herein incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
[0003] The present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods for treating, inhibiting, suppressing, and preventing neurological or neurodegenerative diseases with them.
BACKGROUND OF THE DISCLOSURE
[0004] Many neurodegenerative disorders in patients are difficult to effectively treat, especially where the pathology of a neurodegenerative disorder in a particular patient is not completely understood.
[0005] International Publication No. WO 2016/210372 discloses a method of treating a neurodegenerative disease by administering a PIKFYVE inhibitor.
[0006] There remains a need for effective treatments for many neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). BRIEF SUMMARY OF THE DISCLOSURE
[0007] The present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and their use in the treatment of neurodegenerative disorders.
[0008] In some aspects, the disclosure provides a single stranded ASO that suppresses the expression of PIKFYVE, wherein the ASO has a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the disclosure provides a single stranded ASO that suppresses the expression of PIKFYVE, wherein the ASO has a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804- 1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840- 1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938- 1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072- 2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123- 2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187- 2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0009] The nucleobase sequence of the ASO can comprise up to 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the nucleobase sequence of the ASO can comprise up to 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804- 1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840- 1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938- 1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072- 2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123- 2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187- 2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0010] The ASO can also be any of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376- 420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646- 2321. In some aspects, the ASO can also be any of SEQ ID NOs: 1-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822- 1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236- 2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321. [0011] In some aspects, the disclosure provides an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the disclosure provides an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791- 1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919- 1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985- 1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044- 2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111- 2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166- 2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0012] The oligonucleotide can comprise up to 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the oligonucleotide can comprise up to 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782- 1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026- 2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099- 2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146- 2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0013] In some aspects, at least one internucleoside linkage is a modified intemucleoside linkage, and the modified internucleoside linkage may be a phosphorothioate internucleoside linkage or a phosphodiester intemucleoside linkage. At least one of the nucleosides may also be a modified nucleobase.
[0014] In some aspects, at least one nucleoside of the ASO may be a modified sugar moiety, where that modified sugar moiety can be a bicyclic sugar moiety, or the modified sugar moiety may comprise a 2'-O-methoxyethyl group. In certain aspects, the bicyclic sugar moiety comprises a 4'-CH(R)-O-2' bridge where the R group is, independently, H, C1-12 alkyl, or a protecting group.
[0015] In some aspects, the ASO is a gapmer (e.g., a MOE gapmer), where a gap segment may consist of 8 to 12 linked deoxynucleosides, a 5' wing segment consisting of 3 to 5 linked nucleosides, and a 3' wing segment consisting of 3 to 5 linked nucleosides. In certain aspects, the gap segment may be positioned between the 5' wing segment and the 3' wing segment, where a nucleoside of each wing segment comprises a modified sugar moiety (e.g., one with a 2'-O- methoxy ethyl group).
[0016] In some aspects, the oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the oligonucleotide consists of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782- 1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026- 2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099- 2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146- 2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0017] In some aspects, the disclosure provides a pharmaceutical composition comprising a PIKFYVE ASO of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, and/or excipients. In some aspects, the pharmaceutical composition is suitable for parenteral administration, such as intracerebroventricular injection or intrathecal administration. [0018] In some aspects, the disclosure provides a method of inhibiting, suppressing, or preventing expression of PIKFYVE in a patient (such as one having a neurological or neurodegenerative disease) by administering (for example by, intracerebroventricular injection or intrathecal administration) a PIKFYVE ASO or a pharmaceutical composition described herein (for instance, an effective amount thereof) to the patient.
[0019] In some aspects, the disclosure provides a method of treating a subject having a neurological or neurodegenerative disease by administering a therapeutically effective amount of a PIKFYVE ASO or a pharmaceutical composition described herein. In some aspects, the disease is amyotrophic lateral sclerosis (ALS) (e.g., C9orf72-associated ALS). In some aspects, the disease is frontotemporal dementia (FTD), such as FTD with TDP-43 pathology or FTD with tau pathology. In some aspects, the disease is C9orf72-associated FTD (C9-FTD). In some aspects, the disease is microtubule-associated protein tau (MAPT)-associated FTD (MAPT- FTD), such as FTD with the V337M MAPT mutation.
[0020] In some aspects, the disclosure provides a method of treating a subject having a PIKFYVE disease or disorder by administering a therapeutically effective amount of a PIKFYVE ASO or a pharmaceutical composition described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIGs. 1 A and IB predict skipping of exon 3 of PIKFYVE will generate a stop codon to cause early-termination of the translation of the PIKFYVE protein. represents a stop codon. The sequence shown is taken from Ensembl Database (ENST00000264380).
[0022] FIGs. 2 A and 2B predict skipping of exon 5 of PIKFYVE will make major structural change to the PIKFYVE protein. represents a stop codon. The sequence shown is taken from Ensembl Database (ENST00000264380).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
Definitions
[0024] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” “may” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures.
[0025] The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. [0026] The present disclosure also contemplates other aspects “comprising,” “consisting of’ and “consisting essentially of,” the aspects or elements presented herein, whether explicitly set forth or not.
[0027] As used herein, “unmodified versions” of an ASO/sequence means an ASO/sequence that comprises only DNA or RNA nucleosides connected by phosphodiester linkages.
[0028] As used herein, “2'-deoxynucleoside” means a nucleoside comprising a 2'-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA) and a nucleobase. In some aspects, a 2'-deoxynucleoside may comprise a modified nucleobase and a furanosyl sugar moiety or may comprise an RNA nucleobase (uracil) and a furanosyl sugar moiety.
[0029] As used herein, “2'-substituted nucleoside” means a nucleoside comprising a 2'- substituted sugar moiety. As used herein, “2'-substituted” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.
[0030] As used herein, “antisense molecule” means an oligomeric nucleic acid or oligomeric duplex capable of achieving at least one antisense activity.
[0031] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.
[0032] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0033] As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In some aspects, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In some aspects, the bicyclic sugar moiety does not comprise a furanosyl moiety. As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
[0034] As used herein, “chirally enriched population” means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In some aspects, the molecules are modified oligonucleotides. In some aspects, the molecules are compounds comprising modified oligonucleotides.
[0035] As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); cytosine (C) and guanine (G); and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
[0036] As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.” Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2'-deoxyfuranosyl. Thus, the term “MOE gapmer” indicates a gapmer having a sugar motif of 2'- MOE nucleosides in both wings and a gap of 2'-deoxynucleosides. Unless otherwise indicated, a MOE gapmer may comprise one or more modified internucleoside linkages and/or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications. Table 2 and Table 6, below, provides exemplary MOE-gapmers.
[0037] In some aspects, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.
[0038] In some aspects, modified oligonucleotides comprise or consist of a region having a gapmer motif, which is defined by two external regions or "wings" and a central or internal region or “gap.” The three regions of a gapmer motif include the “5’ wing”, the “gap” and the “3’ wing” which form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3 '-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In some aspects, the sugar moieties within the gap are the same as one another. In some aspects, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In some aspects, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In some aspects, the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).
[0039] In some aspects, the wings of a gapmer comprise 1-5 nucleosides. In some aspects, each nucleoside of each wing of a gapmer is a modified nucleoside.
[0040] In some aspects, the gap of a gapmer comprises 7-12 nucleosides (e.g., 10 nucleosides). In some aspects, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxy nucleoside.
[0041] In some aspects, the gapmer is a deoxy gapmer. In some aspects, the nucleosides on the gap side of each wing/gap junction are unmodified 2'-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In some aspects, each nucleoside of the gap is an unmodified 2'-deoxy nucleoside. In some aspects, each nucleoside of each wing of a gapmer is a modified nucleoside.
[0042] In some aspects, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In some aspects, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In some aspects, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In some aspects, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In some aspects, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In some aspects, each nucleoside of a uniformly modified oligonucleotide comprises the same 2'-modification.
[0043] “Inhibit” as used herein refers to the ability to substantially antagonize, prohibit, prevent, suppress, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of the activity of a particular agent (e.g., infectious agent) or disease.
[0044] As used herein, the term “internucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified internucleoside linkage” means any intemucleoside linkage other than a phosphodiester internucleoside linkage. “Phosphorothioate linkage” is a modified internucleoside linkage in which one of the nonbridging oxygen atoms of a phosphodiester intemucleoside linkage is replaced with a sulfur atom.
[0045] In some aspects, nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage. The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing intemucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates or other alkylphosphonates, phosphoramidates, and phosphorothioates, and phosphorodithioates. Representative non-phosphorus containing intemucleoside linking groups include but are not limited to methylenemethylimino (-CH2- N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-Sitfc-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified intemucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non- phosphorous-containing intemucleoside linkages are well known to those skilled in the art. [0046] Representative intemucleoside linkages having a chiral center include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereo-random intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In some aspects, populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereo-random. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In some aspects, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleoside linkages in a particular, independently selected stereochemical configuration. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In some aspects, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); Chapter 10 of Locked Nucleic Acid Aptamers in Nucleic Acid and Peptide Aptamers: Methods and Protocols v 535, 2009 by Barciszewski et al., editor Gunter Mayerand; and WO 2017/015555. In some aspects, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In some aspects, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Rp) configuration.
[0047] As used herein, “MOE” means methoxy ethyl. “2'-M0E” means a -OCH2CH2OCH3 group at the 2' position of a furanosyl ring.
[0048] A “neurological disease” is any disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, a neurological disease may be a neurodegenerative disease. The neurodegenerative disease may result in motor neuron degeneration, for example. The neurological disease may be amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, or frontotemporal dementia, for example. Further examples of neurological diseases include, but are not limited to, Parkinson’s disease, multiple sclerosis, peripheral myopathy, Rasmussen’s encephalitis, attention deficit hyperactivity disorder, autism, central pain syndromes, anxiety, and/or depression, for example.
[0049] The neurological disease may be associated with aberrant endosomal trafficking. For example, endosomal pathways and endosomes are necessary components for the recycling or breakdown of membrane-bound proteins, trafficking of Golgi-associated proteins, and the extracellular release of proteins in exosomes. These processes aid neurotransmission and drive a balance between recycling and degradation of synaptic vesicles or neurotransmitter receptors, for example.
[0050] The neurological disease may be associated with aberrant lysosome degradation. Alterations in the lysosome degradation may be present in the neurological disease, such as a neurodegenerative disease. Cathepsin imbalance during aging and age-related diseases may provoke deleterious effects on central nervous system (CNS) neurons and lysosomes may be sites for the unfolding and partial degradation of membrane proteins or their precursors that subsequently become expelled from a cell, or are released from dead cells and accumulate as pathological entities.
[0051] A health care professional may diagnose a subject as having a disease associated with motor neuron degeneration by the assessment of one or more symptoms of motor neuron degeneration. To diagnose a neurological disease, a physical exam may be followed by a thorough neurological exam. The neurological exam may assess motor and sensory skills, nerve function, hearing and speech, vision, coordination and balance, mental status, and changes in mood or behavior. Non-limiting symptoms of a disease associated with a neurological disease may be weakness in the arms, legs, feet, or ankles; slurring of speech; difficulty lifting the front part of the foot and toes; hand weakness or clumsiness; muscle paralysis; rigid muscles; involuntary jerking or writing movements (chorea); involuntary, sustained contracture of muscles (dystonia); bradykinesia; loss of automatic movements; impaired posture and balance; lack of flexibility; tingling parts in the body; electric shock sensations that occur with movement of the head; twitching in arm, shoulders, and tongue; difficulty swallowing; difficulty breathing; difficulty chewing; partial or complete loss of vision; double vision; slow or abnormal eye movements; tremor; unsteady gait; fatigue; loss of memory; dizziness; difficulty thinking or concentrating; difficulty reading or writing; misinterpretation of spatial relationships; disorientation; depression; anxiety; difficulty making decisions and judgments; loss of impulse control; difficulty in planning and performing familiar tasks; aggressiveness; irritability; social withdrawal; mood swings; dementia; change in sleeping habits; wandering; and change in appetite.
[0052] Tests may be performed to rule diseases and disorders that may have symptoms similar to those of neurological diseases, measure muscle involvement, assess neuron degeneration. Non-limiting examples of tests are electromyography (EMG); nerve conduction velocity study; laboratory tests of blood, urine, or other substances; magnetic resonance imaging (MRI); magnetic resonance spectroscopy; muscle or nerve biopsy; transcranial magnetic stimulation; genetic screening; x-rays; fluoroscopy; angiography; computed tomography (CT); positron emission tomography; cerebrospinal fluid analysis; intrathecal contrast-enhanced CT scan; electroencephalography; electronystagmography; evoked response; polysomnogram; thermography; and ultrasound. A health care professional may also assess the patient’s family history of diseases associated with motor neuron degeneration and make a diagnosis in part based on a familial history of neurological diseases. A healthcare professional may diagnose a disease associated with neurological disease in a subject after the presentation of one or more symptoms. [0053] Neurodegenerative diseases result in the progressive destruction of neurons that affects neuronal signaling. For example, a neurodegeneration may be amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, Friedreich’s ataxia, Lewy body disease, Parkinson’s disease, spinal muscle atrophy, primary lateral sclerosis, progressive muscle atrophy, progressive bulbar palsy, and pseudobulbar palsy.
[0054] Diseases associated with motor neuron degeneration may be a condition that results in the progressive destruction of motor neurons that interferes with neuronal signaling to the muscles, leading to muscle weakness and wasting. In healthy individuals, upper motor neurons transmit signals from the brain to lower motor neurons in the brain stem and spinal cord, which then transmit the signal to the muscles to result in voluntary muscle activity. The destruction of upper and lower motor neurons affects activity such as breathing, talking, swallowing, and walking, and overtime these functions can be lost. Examples of motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscle atrophy, progressive bulbar palsy, and pseudobulbar palsy. [0055] Neuronal hyperexcitability may occur when receptors for the excitatory neurotransmitter glutamate (glutamate receptors) such as the NMDA receptor and AMPA receptor are over-activated by excess glutamate or by other compounds or neurotransmitters acting on the glutamate receptors. Excitotoxicity may result from neuronal hyperexcitability. Exci totoxi city is the pathological process by which nerve cells are damaged or killed by excessive stimulation. The excessive stimulation allows high levels of calcium ions (Ca2+) to enter the cell. Ca2+ influx into cells activates a number of enzymes, including phospholipases, endonucleases, and proteases such as calpain. These enzymes can damage cell structures such as components of the cytoskeleton, membrane, and DNA.
[0056] Neuronal hyperexcitability may be involved in spinal cord injury, stroke, traumatic brain injury, hearing loss (through noise overexposure or ototoxicity), epilepsy, painful neuropathies, attention deficit hyperactivity disorder, autism, central pain syndromes, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, schizophrenia, Rasmussen’s encephalitis, Huntington’s disease, alcoholism or alcohol withdrawal and especially over-rapid benzodiazepine withdrawal, and also Huntington's disease. Other common conditions that cause excessive glutamate concentrations around neurons are hypoglycemia. Blood sugars are the primary glutamate removal method from inter-synaptic spaces at the NMDA and AMPA receptor site.
[0057] As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
[0058] As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. As used herein, an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase or modified nucleobase. A “5 -methylcytosine” or “mC” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification. [0059] In some aspects, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In some aspects, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In some aspects, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
[0060] In some aspects, modified nucleobases are selected from: 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In some aspects, modified nucleobases are selected from: 2- aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (- OC-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8- substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5- halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N- benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3- diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3- diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.
[0061] As used herein, “nucleoside” means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are presented between those that are linked).
[0062] As used herein, “oligomeric compound” means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “singled stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.” [0063] As used herein, “oligonucleotide” means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. The internucleoside linkages may be any described herein. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.
[0064] “PIKFYVE”, also known in the art as “phosphatidylinositol-3-phosphate 5-kinase type III” or “PIPKIII”, is a FYVE finger-containing phosphoinositide kinase encoded by the PIKFYVE gene. PIKFYVE is a highly evolutionarily conserved lipid kinase and also has protein kinase activity, which regulates endomembrane homeostasis and plays a role in the biogenesis of endosome carrier vesicles from early endosomes. PIKFYVE-mediated conversion of PI3P to PI(3,5)P2 blocks recruitment of the protein EEA1. The recruitment is blocked, because PIP3 is needed to form a platform with RAB5 that enables anchoring of EEA1 to early endosomes. EEA1 then drives fusion with endocytic and other endosomal vesicles.
[0065] As used herein a “PIKFYVE disease or disorder” includes lysosomal degradation diseases and disorders mediated by PIKFYVE. For example, the a PIKFYVE disease or disorder includes, but is not limited to, amyloid diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis and chronic hemodialysis-related amyloid), multiple sclerosis, and an MPS disorder (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). In some aspects, the diseases are autoimmune disorders (such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, Ankylosing spondylitis, psoriasis, psoriatic arthritis, adult still disease, Becet syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease), or CNS disorders (such as spongiform encephalopathies (Creutzfeld- Jakob, Kuru, Mad Cow)). The compositions and methods of the disclosure can be used to treat individuals with lysosomal storage diseases comprising administering to a subject in need of treatment a therapeutically effective amount of a PIKfyve ASO or pharmaceutical composition described herein. In some aspects, the ASOs and compositions of the disclosure decrease or inhibit the activity of PIKfyve and alters the biogenesis, function or dynamics of the endosomal or lysosomal systems in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases. In some aspects, the ASOs and compositions target, decrease or inhibit the activity of PIKfyve thus altering the biogenesis, functions, or dynamics of the endoplasmic reticulum or Golgi apparatus in a way that reduces the abundance of the material abnormally stored in the lysosome in lysosomal storage diseases. In some aspects, the disease is a neurological disorder.
[0066] As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. The superscript prime symbol (') is used to describe the numbering of a sugar in a nucleoside or nucleotide (the nucleobase positions are numbered without the prime). When describing the sugar only, the prime symbol is not used. As used herein, “unmodified sugar moiety” means a 2-0H(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moi eties have one hydrogen at each of the 1, 3, and 4 positions, an oxygen at the 3 position, and two hydrogens at the 5 position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In some aspects, a modified furanosyl sugar moiety is a 2-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and nonbicyclic sugars.
[0067] In some aspects, modified sugar moieties are nonbicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2, 4, and/or 5 positions. In some aspects, one or more non-bridging substituent of nonbicyclic modified sugar moieties is branched. Examples of 2-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2-F, 2-OCHs (“OMe” or “O-methyl”), and 2- O(CH2)2OCH3 ("MOE"). In some aspects, 2-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O-Ci-10 alkoxy, O-Ci-10 substituted alkoxy, O-Ci- 10 alkyl, O-Ci-10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O- alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O- aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), where each Rm and R„ is, independently, H, an amino protecting group, or substituted or unsubstituted C1-10 alkyl, and the 2-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of d'substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), and alkyl. Examples of 5-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5-methyl (R or S), 5-vinyl, and 5- methoxy. In some aspects, non-bicyclic modified sugar moieties comprise more than one nonbridging sugar substituent, for example, 2-F-5-methyl sugar moieties and the like.
[0068] In some aspects, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a nonbridging 2'-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)- N(Rm)(Rn)), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-10 alkyl.
[0069] In some aspects, a 2'-substituted nucleoside nonbicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 (“NMA”).
[0070] In some aspects, a 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a nonbridging 2'-substituent group selected from: F, OCH3, and OCH2CH2OCH3.
[0071] Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In some aspects, the bicyclic sugar moiety comprises a bridge between the 4 and the 2 furanose ring atoms. Examples of such 4 to 2 bridging sugar substituents include but are not limited to: 4-CEE-2, 4-(CH2)2-2, 4- (CH2)3-2, 4-CH2-O-2 (“LNA”), 4-CH2-S-2, 4-(CH2)2-O-2 (“ENA”), 4-CH(CH3)-O-2 (referred to as “constrained ethyl” or “cEf ’), 4-CH2-O-CH2-2, 4-CH2-N(R)-2, 4-CH(CH2OCH3)-O-2 (“constrained MOE” or “cMOE”) and analogs thereof, 4-C(CH3)(CH3)-O-2 and analogs thereof, 4-CH2-N(OCH3)-2 and analogs thereof, 4-CH2-O-N(CH3)-2, 4-CH2-C(H)(CH3)-2, 4-CH2- C(=CH2)-2 and analogs thereof, 4-C(RaRb)-N(R)-O-2, 4-C(RaRb)-O-N(R)-2, 4-CH2-O-N(R)-2, and 4-CH2-N(R)-O-2, wherein each R, Ra, and Rb, is, independently, H, a protecting group, or Ci- 12 alkyl.
[0072] In some aspects, such 4 to 2 bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(Ra)=C(Rb)-, -C(Ra)=N-, - C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)X-, and - N(Ra)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-12 alkyl, substituted C1-12 alkyl, C1-12 alkenyl, substituted C2-12 alkenyl, C2-12 alkynyl, substituted C2-12 alkynyl, C5-20 aryl, substituted C5-20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-7 alicyclic radical, substituted C5-7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N3, COOJi, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=O)-Ji); and each Ji and J2 is, independently, H, C1-12 alkyl, substituted C1-12 alkyl, C2-12 alkenyl, substituted C2-12 alkenyl, C2-12 alkynyl, substituted C2-12 alkynyl, C5-20 aryl, substituted C5-20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-12 aminoalkyl, substituted C1-12 aminoalkyl, or a protecting group.
[0073] Additional bicyclic sugar moieties are known in the art, see, for example: Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek etal., J. Org. Chem., 2006, 71, 7731- 7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 20017, 129, 8362-8379; Wengel et a., U.S. Pat. No. 7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al. U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727. [0074] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some aspects, the subject may be a human or a non-human. In some aspects, the subject or patient is a human. The subject or patient may be undergoing other forms of treatment. In some aspects, the patient has a neurological disease due to a mutation in the C9ORF72 gene (for instance, the patient may be haploinsufficient for the C9ORF72 gene (e.g., one which results in a 50% or greater reduction in C9ORF72 protein activity), or the C9ORF72 gene may comprises a GGGGCC repeat expansion (such as a (GGGGCC)n (SEQ ID NO: 1565) hexanucleotide expansion in C90RF72)). The variable “n” can be at least 30.
[0075] A “therapeutically effective amount,” or “effective dosage” or “effective amount” as used interchangeably herein unless otherwise defined, means a dosage of a drug effective for periods of time necessary, to achieve the desired therapeutic result. An effective dosage may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, mammal, or human, such as reducing and/or inhibiting the function of a receptor. A therapeutically effective amount may be administered in one or more administrations (e.g., the agent may be given as a preventative treatment or therapeutically at any stage of disease progression, before or after symptoms, and the like), applications or dosages and is not intended to be limited to a particular formulation, combination or administration route. It is within the scope of the present disclosure that the drug may be administered at various times during the course of treatment of the subject. The times of administration and dosages used will depend on several factors, such as the goal of treatment (e.g., treating v. preventing), condition of the subject, etc. and can be readily determined by one skilled in the art.
[0076] As used herein, the term “treat” or “treating” a subject, refers to administering a composition or agent described herein to the subject, such that at least one symptom of a disease or disorder is healed, alleviated, relieved, altered, remedied, reduced, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, reduce, ameliorate, and/or improve one or more symptoms associated with a disease or disorder. The treatment may inhibit deterioration or worsening of a symptom associated with the disease or disorder.
[0077] The herein described methods of treatment may comprise administering to a subject in need thereof a composition comprising an effective amount of one or more antisense oligonucleotides that treats neurological diseases by inhibiting or suppressing PIKFYVE expression. The one or more antisense oligonucleotides may decrease or inhibit neurodegeneration. The one or more antisense oligonucleotides may decrease neuronal hyperexcitability. Reducing PIKFYVE mRNA and PIKFYVE protein levels suppresses neurodegeneration driven by toxic TDP-43 aggregates, DPR aggregates (for example, in C9ORF72-ALS patients), and by promoting the retention of TDP-43 in the nucleus. Delivery of the ASOs targeting PIKFYVE mRNA described herein reduces PIKFYVE protein levels.
[0078] The composition may inhibit kinase activity by inhibiting expression of a kinase. The composition may inhibit PIKFYVE kinase activity or expression. The one or more antisense oligonucleotides can be combined with small molecule therapeutic agents (such as apilimod and/or YM201636).
[0079] The disclosure provides oligonucleotides (modified or unmodified) that can be used to modulate PIKFYVE expression. Table 1 provides (5' to 3') generic sequence of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure. Table 6 provides (5' to 3') modified sequence of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure. Unmodified sequences of Table 6 are additionally envisioned as generic sequences of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the disclosure. The ASO sequences of SEQ ID NOs: 1-49 are predicted to skip exon 3 of PIKFYVE efficiently, and generate a stop codon to cause early- termination of PIKFYVE translation , as shown in FIGs. 1 A and IB. The ASO sequences of SEQ ID NOs: 50-80 are predicted to skip exon 5 of PIKFYVE efficiently, and make a major structural change to the protein rendering it non-functional, as shown in FIGs. 2A and 2B. The ASO sequences SEQ ID NOs: 81-328, 330-373, 376-420, 423-479 have low acute neurotoxicity scores as determined by the method described in Hagedorn et al., Nucleic Acid Therapeutics, 2022, 32(3): 151-162 (DOI: 10.1089/nat/2021/0071). The ASO sequences SEQ ID NOs: 307, 308, 330, 424, 480-540, 542-619, 621-685, 688-782, and the unmodified versions of SEQ ID NOs: 1646-2321 have low numbers of predicted off-target binding sites as determined by the GGGenome search tool using 2 mismatches/gaps.
TABLE 1
[0080] In some aspects, the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 in Table 1; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspect, the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 in Table 1 or and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782- 1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-
2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-
2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-
2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234,
2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321. [0081] In some aspects, the modified oligonucleotide is at least 80% to 100% (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100%; or any numerical range or value between any of the foregoing values) identical to any of the sequences comprising or consisting of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. In some aspects, the modified oligonucleotide is at least 80% to 100% (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100%; or any numerical range or value between any of the foregoing values) identical to any of the sequences comprising or consisting of SEQ ID NOs: 1-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822- 1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236- 2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
[0082] The sequences provided in Table 1 and the unmodified versions of the sequences of Table 6 can be used to design antisense molecules for inhibition of PIKFYVE expression. For example, gapmer oligonucleotides can be designed using the sequences in Table 1 and the unmodified versions of the sequences of Table 6, and can comprise a 5'-wing of about 3-5 nucleotides, a 3'-wing of about 3-5 nucleotides and a gap region comprising 8-12 consecutive deoxyribonucleosides of any one of the sequences of Table 1 and the unmodified versions of the sequences of Table 6.
[0083] In some aspects, an oligonucleotide of the disclosure comprises a gapmer having a gap segment of at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 in Table 1; and the unmodified versions of SEQ ID NOs: 1646-2321; flanked by a 5' and 3' wing segments, wherein the gap segment is located between the 5' and 3' wing segments and wherein each of the wing segments comprises a modified sugar. In some aspects, the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length. In some aspects, an oligonucleotide of the disclosure comprises a 5' wing segment comprising modified sugars and having the nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542- 619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321, followed by a 3' wing segment comprising modified sugars and having the nucleobase sequence of the last 3-5 nucleobases of the same sequence corresponding to SEQ ID NOs: 1-782, for example 1-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782; and the unmodified versions of SEQ ID NOs: 1646-2321. Table 2 and Table 6 provide MOE gapmers of the disclosure.
[0084] In some aspect, an oligonucleotide of the disclosure comprises a gapmer having a gap segment of at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425- 498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 in Table 1 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822- 1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236- 2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321; flanked by a 5' and 3' wing segments, wherein the gap segment is located between the 5' and 3' wing segments and wherein each of the wing segments comprises a modified sugar. In some aspects, the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length. In some aspects, an oligonucleotide of the disclosure comprises a 5' wing segment comprising modified sugars and having the nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804- 1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840- 1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938- 1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072- 2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123- 2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187- 2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791- 1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919- 1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985- 1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044- 2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111- 2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166- 2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321, followed by a 3' wing segment comprising modified sugars and having the nucleobase sequence of the last 3-5 nucleobases of the same sequence corresponding to SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425- 498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791- 1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919- 1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985- 1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044- 2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111- 2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166- 2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321. Table 2 and Table 6 provide MOE gapmers of the disclosure.
[0085] The 5' and/or 3' wings can comprise the following chemistries: 2'-OMe, 2'-M0E, LNA or DNA, by themselves or used in combination with one another. The backbone linkage of the 5' and/or 3' wings can be phosphorothioate or a mixture of phosphodiester and phosphorothioate. Linkages in the gap region can be phosphorothioate.
[0086] In some aspects, the oligonucleotide is single stranded. In some aspects, the oligonucleotide comprises or is complexed with a moiety that neutralizes charge on the oligonucleotide to promote uptake and transfer across a cell membrane.
[0087] In some aspects, the ASO sequences of SEQ ID NOs: 1-80 in Table 1 have the following 5-8-5 motif: 2MOE*2MOE-2MOE-2MOE-2MOE-N*N*N*N*N*N*N*N*2MOE- 2MOE-2MOE*2MOE*2MOE where (i) 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E), (ii) N is a nucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage. Table 2 below shows this motif on SEQ ID NOs: 1-80 (here SEQ ID NOs: 783-862). In some aspects, ASO sequences of SEQ ID NOs: 783-831 skip exon 3 of PIKFYVE efficiently. In some aspects, ASO sequences of SEQ ID NOs: 832-862 skip exon 5 of PIKFYVE efficiently.
[0088] In some aspects, the ASO sequences of SEQ ID NOs: 1-782, for example 81-328, 330- 373, 376-420, 423-540, 542-619, 621-685, 688-782, in Table 1 and t the unmodified versions of SEQ ID NOs: 1646-2321 have the following 5-10-5 motif: 2MOE*2MOE-2MOE-2MOE- 2MOE-N*N*N*N*N*N*N*N*N*N*2MOE-2MOE-2MOE*2MOE*2MOE where (i) 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E), (ii) N is a nucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage. Table 2 below shows this motif on SEQ ID NOs: 1-782, for example 81-328, 330-373, 376-420, 423-540, 542-619, 621-685, 688-782 (here SEQ ID NOs: 783-1564, for example 863- 1110, 1112-1155, 1158-1202, 1205-1322, 1324-1401, 1403-1467, 1470-1564).
[0089] In some aspects, the ASO sequences of SEQ ID NOs: 81-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, in Table 1 and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782- 1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026- 2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099- 2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146- 2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321 have the following 5-10-5 motif: 2MOE*2MOE-2MOE-2MOE-2MOE-
N*N*N*N*N*N*N*N*N*N*2MOE-2MOE-2MOE*2MOE*2MOE where (i) 2M0E is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-M0E), (ii) N is a nucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage.
[0090] Table 2: The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Gapmer design: 5'- five 2'-methoxyethylribose nucleotides - ten DNA nucleotides - five 2'- methoxy ethylribose nucleotides - 3'; Capital letters are 2'-methoxyethylribose nucleosides; lower case are DNA nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
TABLE 2
[0091] In some aspects, the ASO sequences of SEQ ID NOs: 1-49 in Table 1 have the following motif: 2MOE*2MOE-2MOE-2MOE-2MOE-2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE-2MOE-2MOE*2MOE*2MOE where (i) 2MOE is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) the asterisk (*) refers to a phosphorothioate linkage, and (iii) the dash (-) refers to a phosphodiester linkage. Table 3 below shows this motif on SEQ ID NOs: 1-49 (here SEQ ID NOs: 1566-1614). In some aspects, ASO sequences of Table 3 skip exon 3 of PIKFYVE efficiently. In some aspects, an ASO of a sequence set forth in SEQ ID NOs: 1-49 skips exon 3 of PIKFYVE efficiently.
[0092] Table 3: The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Design: 5'- eighteen 2'-methoxyethylribose nucleotides - 3'; Capital letters are 2'- methoxyethylribose nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
TABLE 3
[0093] In some aspects, the ASO sequences of SEQ ID NOs: 50-80 in Table 1 have the following motif: 2MOE*2MOE-2MOE-2MOE-2MOE-2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE *2MOE-2MOE-2MOE*2MOE*2MOE where (i) 2MOE is a nucleobase with a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) the asterisk (*) refers to a phosphorothioate linkage, and (iii) the dash (-) refers to a phosphodiester linkage. Table 4 below shows this motif on SEQ ID NOs: 50-80 (here SEQ ID NOs: 1615-1645). In some aspects, ASO sequences of Table 4 skip exon 5 of PIKFYVE efficiently. In some aspects, an ASO of a sequence set forth in SEQ ID NOs: 50-80 skip exon 5 of PIKFYVE efficiently.
[0094] Table 4: The Sequence of Bases in PIKFYVE Antisense Oligonucleotides - (ASOs). (Design: 5'- eighteen 2'-methoxyethylribose nucleotides - 3'; Capital letters are 2'- methoxyethylribose nucleosides; asterisks (*) are phosphorothioate linkages; linkages which do not have an asterisk are phosphodiester linkages) (Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated).
TABLE 4
[0095] The PIKFYVE kinase antisense or inhibitory nucleic acids of the disclosure can inhibit the expression and thus the activity associate with PIKFYVE. The PIKFYVE kinase antisense or inhibitory nucleic acids can include any combination of the oligonucleotides set forth in Tables 1, 2, 3, 4, 5, and 6 and sequences that are 98%-99% identical thereto.
[0096] The PIKFYVE ASOs described herein, such as SEQ ID NOs: 783-1564, for example 783-1110, 1112-1155, 1158-1202, 1205-1322, 1324-1401, 1403-1467, 1470-1564, and 1566- 2321, suppress PIKFYVE mRNA expression with minimal off-target binding.
[0097] In some aspects, the PIKFYVE ASOs described herein, such as SEQ ID NOs: 783- 1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, and 1566-2321, for example SEQ ID NOs: 863-1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563; suppress PIKFYVE mRNA expression with minimal off-target binding.
[0098] Methods of treatment may include any number of modes of administering a disclosed composition. Modes of administration may include aqueous, lipid, oily or other solutions, solutions in simulated cerebrospinal fluid, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions and the like. Typically, an ASO of the disclosure will be administered directly to the CNS of the subject. Accordingly, the formulation or composition will be sterile and more preferably be suitable for injection. The following formulations and methods are merely exemplary and are in no way limiting.
[0099] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be presented in unit-dose or multidose sealed containers, such as ampules and vials, and may be stored as liquids or in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulation may be provided in a pre-filled syringe.
[0100] Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed one or more antisense or inhibitory nucleic acids and compositions. Sequential administration includes administration before or after the disclosed one or more antisense or inhibitory nucleic acids or compositions. In some aspects, the additional therapeutic agent or agents may be administered in the same composition as the disclosed one or more antisense or inhibitory nucleic acids. In some aspects, there may be an interval of time between administration of the additional therapeutic agent and the disclosed one or more antisense or inhibitory nucleic acids. In some aspects, administration of an additional therapeutic agent with a disclosed one or more antisense or inhibitory nucleic acids may allow lower doses of the other therapeutic agents and/or administration at less frequent intervals. When used in combination with one or more other active ingredients, the one or more antisense or inhibitory nucleic acids of the disclosure and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions of the disclosure include those that contain one or more other active ingredients, in addition to one or more antisense or inhibitory nucleic acids of the disclosure. The above combinations include combinations of one or more antisense or inhibitory nucleic acids of the disclosure not only with one other active compound, but also with two or more other active compounds. For example, the compound of the disclosure may be combined with a variety of drugs to treat neurological diseases. The antisense oligonucleotide may be covalently linked to another oligonucleotide, such as one with a target other than PIKFYVE. The antisense oligonucleotide may be covalently linked to an antibody. [0101] The disclosed one or more antisense or inhibitory nucleic acids can be combined with the following, but are not limited, anticholinergic drugs, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, pain medications, and/or stimulants.
Additional types of therapy and treatment include, but are not limited to digital communication devices, feeding tubes, mechanical ventilation, nutritional support, deep brain stimulation, occupational therapy, physical therapy, and/or speech therapy.
[0102] The disclosed composition(s) may be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which may be a human or non-human). The pharmaceutical compositions may comprise a carrier (e.g., a pharmaceutically acceptable carrier). Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the composition of the present disclosure.
[0103] The pharmaceutical compositions may include a therapeutically effective amount or a prophylactically effective amount of the antisense oligonucleotide. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of one or more antisense or inhibitory nucleic acids of the disclosure are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[0104] The pharmaceutical compositions may include one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as releasing agents, coating agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.
[0105] The route by which the disclosed one or more antisense or inhibitory nucleic acids are administered, and the form of the composition will dictate the type of carrier to be used.
[0106] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal, intracerebroventricular, or intraventricular, administration. In some aspects, the antisense or inhibitory nucleic acid is administered by intravenous, intraperitoneal, or as a bolus injection or administered directly into the target organ. In some aspects, the antisense or inhibitory nucleic acid is administered intrathecally or intra-cerebroventricular as a bolus injection.
[0107] Carriers for systemic administration typically include at least one of solvent, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.
[0108] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol.
[0109] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%. [0110] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as com starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.
[OHl] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic composition is typically about 0.1 to about 10%.
[0112] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.
[0113] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%. [0114] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.
[0115] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.
[0116] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.
[0117] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.
[0118] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.
[0119] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.
[0120] Compositions and formulations for parenteral, intrathecal, intra-cerebroventricular, or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. For example, an intrathecal cerebrospinal fluid (CSF) catheter can be used to deliver antisense formulations of the disclosure. The catheter can be inserted at the L3 or L4 vertebrae. The distal tip of the catheter extends within the intrathecal space to approximately the LI vertebrae. Antisense oligonucleotides are dissolved in saline, are sterilized by filtration, and are administered at 0.33 ml/min in a 1.0 ml volume followed by a 0.5 ml sterile water flush. Total infusion time is 4.5 min.
[0121] Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.
[0122] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the medicament. Techniques and compositions for making dosage forms useful in the methods of this disclosure are described in the following references: Modem Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).
[0123] In vivo testing of candidate antisense or inhibitory nucleic acids may be conducted by means known to one of ordinary skill in the art. For example, the candidate one or more antisense or inhibitory nucleic acids may be administered to a mammal, such as a mouse or a rabbit. The mammal may be administered, by any route deemed appropriate, a dose of a candidate antisense or inhibitory nucleic acids. Conventional methods and criteria can then be used to monitor animals for signs of reduction or improvement of motor neuron activity and/or expression or activity of PIKFYVE gene or protein, respectively. If needed, the results obtained in the presence of the candidate antisense or inhibitory nucleic acids can be compared with results in control animals that are not treated with the candidate antisense or inhibitory nucleic acids. Dosing studies may be performed in, or in conjunction with, the herein described methods for identifying one or more antisense or inhibitory nucleic acids capable of treating a neurological disease and/or any follow-on testing of candidate antisense or inhibitory nucleic acids in vivo. One of skill in the art of medicine may determine the appropriate dosage of one or more antisense or inhibitory nucleic acids. The dosage may be determined by monitoring the subject for signs of disease inhibition or amelioration. The dosage may be increased or decreased to obtain the desired frequency of treatment. The toxicity and efficacy of one or more antisense or inhibitory nucleic acids may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. determining the lethal dose to 50% of the population (LD50) and the dose therapeutically effective in 50% of the population (ED50). The dose ratio of LD50/ED50 is the therapeutic index and, indicating the ratio between the toxic and therapeutic effects. A delivery system may be designed to help prevent toxic side effects, by delivering the one or more antisense or inhibitory nucleic acids to specific targets, e.g., delivered specifically to motor or central nervous system neurons. The optimal dose of the one or more antisense or inhibitory nucleic acids may be determined based on results of clinical electrophysiology or electromyography to analyze excitability in peripheral nerves, for example.
[0124] The dosage for use in humans may be determined by evaluating data obtained from animal studies and cell culture assays. The preferred dosage will have little or no toxicity and include the ED50. The dosage may vary depending on the dosage form and route of administration. For any antisense or inhibitory nucleic acid used in the methods described herein, the dosage may be estimated initially in cell culture. A dose may be formulated in animal models that includes the concentration of the test compound which achieves a half maximal inhibition of symptoms (LD50) as determined in cell culture. Such information obtained from cell cultures and animal models may be used to more accurately determine useful doses in humans.
[0125] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
EXAMPLES
[0126] Small molecule inhibitors of PIKFYVE kinase and antisense oligonucleotides (ASOs) that suppress PIKFYVE expression can prevent the degeneration of human and mouse neurons that carry a mutation in the C9ORF72 gene that leads to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
[0127] ASOs are an attractive therapeutic option for neurodegenerative diseases because of their ease of delivery to the central nervous system and their relatively low exposure to the periphery. These properties maximize target engagement in the central nervous system and minimize undesired target engagement or off-target effects in the periphery.
[0128] The disclosure provides novel antisense oligonucleotide (ASO) sequences targeting the PIKFYVE gene that can suppress PIKFYVE expression in human cells. PIKFYVE ASOs can also rescue the survival of motor neurons derived from sporadic ALS patients. Moreover, PIKFYVE ASOs can lower levels of neurotoxic dipeptide repeat protein aggregates derived from the C9ORF72 repeat expansion in vivo in mice.
Example 1
[0129] To identify ASO sequences that suppress PIKFYVE expression in human cells, ASOs are designed (see Tables 2, 3, 4, and 6) and are synthesized as MOE gapmers, which contains sugar and linkage modifications that increase nuclease resistance and melting temperature while maintaining the ability to be used as a substrate of RNase H. The ability of each ASO to suppress PIKFYVE RNA levels is tested by transfecting them into human embryonic kidney 293T cells with Lipofectamine 2000 at a concentration of 100 nM and measuring PIKFYVE expression 7 days after transfection. A control (NCASO) is used. The relative PIKFYVE expression shown is an average of three technical replicates and values are calculated by normalizing to a GAPDH control.
Example 2
[0130] In the study, neonate transgenic VPIKFYVE BAC mice receive 25 pg of negative control ASO or test compound by intracerebroventricular (ICV) injection at Pl (postnatal day 1) and tissue samples are collected 14 days post-treatment.
Example 3
[0131] An in silico study of the ASOs of SEQ ID NOs. 863-1261 was conducted to determine the ASO’s acute neurotoxicity score of each ASO. The acute neurotoxicity score was generated using the method described in Hagedorn et al 2022. (DOI: 10.1089/nat/2021/0071). ASOs of SEQ ID NOs. 863-1261 were determined to have low acute neurotoxicity scores. The results of the study are shown below in Table 5. TABLE 5
[0132] In some aspects, an ASO of a sequence set forth in SEQ ID NOs: 81-306, 310-328, 332- 373, 376-420, 423, and 425-479, comprise a low acute neurotoxicity score. Example 4
[0133] Suppression of off-target genes, such as CNTN5, was predicted in silico for various ASOs described herein. An in silico study of various ASOs was conducted to determine the number of off-target binding sites of various ASOs. The number of off-target binding sites was generated using GGGenome search tool using 2 mismatches/gaps. The results of the study are shown below in Table 6.
[0134] The ASO’s of Table 6 have the following Gapmer design: 5'- five 2'- methoxy ethylribose nucleotides - ten DNA nucleotides - five 2'-methoxy ethylribose nucleotides - 3'; wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, sixteenth, and seventeenth linkages are phosphodiester linkages (the same Gapmer design as Table 2). Note that the following table provides 2'MOE wings; however, alternative wings comprising 2'-0Me or LNA (locked nucleic acid) are contemplated.
Additionally note that the following table provides Gapmer ASOs, but ASOs with 20 DNA nucleotides (no 2'MOE wings) and a complete phospodiester backbone (unmodified versions) of the SEQ IDs of Table 6 are contemplated.
TABLE 6 - I l l -
[0135] In some aspects, an ASO of a sequence set forth in SEQ ID NOs: 480-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996- 1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179- 2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321 comprise a low number of off-target binding sites. [0136] The disclosure provides ASOs that suppress PIKFYVE expression in human cells. The accompanying data is consistent with these ASOs ability to prevent, inhibit, or suppress neurodegeneration in ALS and FTD patients.
[0137] The foregoing description and drawings should be considered as illustrative only of the principles of the disclosure. The disclosure is not intended to be limited by the aspects and may be implemented in a variety of ways that will be clear to one of ordinary skill in the art.
Numerous applications of the disclosure will readily occur to those skilled in the art. Therefore, it is not desired to limit the disclosure to the specific examples disclosed or the exact construction and operation shown and described. Rather, all suitable modifications and equivalents may be resorted to, falling within the scope of the disclosure. All references cited herein are incorporated by reference.

Claims

1. A single stranded antisense oligonucleotide that suppresses the expression of a PIKFYVE, wherein the antisense oligonucleotide comprises a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences set forth in SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895- 1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a nucleobase sequence set forth in any one of SEQ ID NOs: 1-306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955- 1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270- 2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
3. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide has 18 to 20 linked nucleosides.
4. The antisense oligonucleotide of any of the preceding claims, wherein at least one internucleoside linkage is a modified internucleoside linkage.
5. The antisense oligonucleotide of claim 4, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
6. The antisense oligonucleotide of claim 4, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.
7. The antisense oligonucleotide of any of the preceding claims, wherein at least one internucleoside linkage is a phosphodiester internucleoside linkage.
8. The antisense oligonucleotide of claim 7, wherein at least one internucleoside linkage is a phosphorothioate linkage and at least one intemucleoside linkage is a phosphodiester linkage.
9. The antisense oligonucleotide of any of the preceding claims, wherein at least one nucleoside comprises a modified nucleobase.
10. The antisense oligonucleotide of claim 9, wherein the modified nucleobase is a 5- methylcytosine.
11. The antisense oligonucleotide of any of the preceding claims, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.
12. The antisense oligonucleotide of claim 11, wherein the modified sugar moiety comprises a 2'-O-methoxy ethyl group.
13. The antisense oligonucleotide of any of the preceding claims, wherein the antisense oligonucleotide is a gapmer.
14. The antisense oligonucleotide of claim 13, wherein the antisense oligonucleotide comprises: a gap segment consisting of 8 to 12 linked deoxynucleosides; a 5' wing segment consisting of 3 to 5 linked nucleosides; and a 3' wing segment consisting of 3 to 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein a nucleoside of each wing segment comprises a modified sugar moiety.
15. The antisense oligonucleotide of claim 14, wherein each nucleoside of each wing segment comprises a modified sugar moiety.
16. The antisense oligonucleotide of claim 14, wherein the nucleosides making up each wing segment comprise at least two different modified sugar moieties.
17. The antisense oligonucleotide of claim 14, wherein the nucleosides making up each wing segment comprise the same modified sugar moiety.
18. The antisense oligonucleotide of claim 15, wherein the modified sugar moiety comprises a 2'-O-methoxy ethyl group.
19. The antisense oligonucleotide of any of the preceding claims, wherein the antisense oligonucleotide comprises 15 to 50 nucleosides.
20. The antisense oligonucleotide of any of the preceding claims, wherein the antisense oligonucleotide comprises a nucleobase sequence that comprises at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences set forth in SEQ ID NOs: 783-1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382- 1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708- 1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747- 1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270- 2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
21. The antisense oligonucleotide of any of the preceding claims, wherein the antisense oligonucleotide comprises the sequence set forth in any of SEQ ID NOs: 783-1088, 1092- 1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911- 1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955- 1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270- 2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
22. A pharmaceutical composition comprising the antisense oligonucleotide of any one of the preceding claims, and a pharmaceutically acceptable carrier, diluent and/or excipient.
23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for parenteral delivery.
24. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for intracerebroventricular injection.
25. A method of treating a subject having a neurological or neurodegenerative disease in need thereof comprising administering a therapeutically effective amount of the antisense oligonucleotide of any one of claims 1-21 or the pharmaceutical composition of any one of claims 22-24.
26. The method of claim 25, wherein the neurological disease is associated with neuronal hyperexcitability.
27. The method of claim 25, wherein the neurological disease is associated with aberrant endosomal trafficking.
28. The method of claim 25, wherein the neurological disease is associated with aberrant lysosomal trafficking.
29. The method of claim 25, wherein the neurological disease is selected from the group consisting of familial and sporadic amyotrophic lateral sclerosis (ALS), familial and sporadic frontotemporal dementia (FTD), progressive supranuclear palsy, Alzheimer's disease, chronic traumatic encephalopathy, Parkinson's disease, Charcot Marie Tooth 2A and 4B, Huntington's disease, dementia, transmissible spongiform encephalopathy, spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxias, and Creutzfeldt- Jakob disease.
30. The method of claim 25, wherein the neurological disease is familial amyotrophic lateral sclerosis.
31. The method of claim 25, wherein the neurological disease is sporadic amyotrophic lateral sclerosis.
32. The method of claim 25, wherein the neurological disease is familial frontotemporal dementia.
33. The method of claim 25, wherein the neurological disease is sporadic frontotemporal dementia.
34. The method of claim 25, wherein the neurological disease is frontotemporal dementia with TDP-43 pathology.
35. The method of claim 25, wherein the neurological disease is frontotemporal dementia with tau pathology.
36. The method of any one of claims 25-35, wherein the subject is haploinsufficient for the C9ORF72 gene.
37. The method of any one of claims 25-35, wherein the subject has a GGGGCC repeat expansion in C90RF72.
38. The method of any one of claims 25-35, wherein the subject has a (GGGGCC)n (SEQ ID NO: 1565) hexanucleotide expansion in C90RF72, where n is at least 30.
39. The method of any one of claims 25-35, wherein the subject has C9orf72-associated frontotemporal dementia.
40. The method of any one of claims 25-35, wherein the subject has microtubule-associated protein tau (MAPT)-associated frontotemporal dementia.
41. The method of claim 40, wherein the patient has the V337M MAPT mutation.
42. A method of inhibiting or suppressing expression of PIKFYVE in a patient having a neurological or neurodegenerative disease comprising administering an effective amount of the antisense oligonucleotide of any one of claims 1-21 or the pharmaceutical composition of any one of claims 22-24.
43. An oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences set forth in SEQ ID NOs: 1- 306, 310-328, 332-373, 376-420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, 783-1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791- 1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270- 2289, 2291-2300, 2302, 2304-2316, and 2319-2321, and the unmodified versions of SEQ ID Nos: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762- 1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321.
44. The antisense oligonucleotide of claim 1, which comprises the sequence set forth in SEQ ID NOs: 1-80.
45. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide suppresses the expression of a PIKFYVE by at least 80%.
46. A single stranded antisense oligonucleotide that suppresses the expression of a PIKFYVE, wherein the antisense oligonucleotide has a nucleobase sequence that comprises the sequence set forth in any of SEQ ID NOs: 1-306, 310-328, 332-373, 376- 420, 423, 425-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733- 734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762- 1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895-1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971- 1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321, wherein the first 3-5 nucleosides at the 5’ end (“5’ wing segment”) comprise modified sugars, the last 3-5 nucleosides at the 3’ end (“3’ wing segment”) comprise modified sugars, and the remaining nucleosides comprise a gap segment.
47. The antisense oligonucleotide of claim 46, wherein the modified sugars comprise 2'-OMe, 2'-M0E, LNA, or any combination thereof.
48. The antisense oligonucleotide of claim 47, wherein at least one of the modified sugars comprises 2'-OMe.
49. The antisense oligonucleotide of claim 47, wherein at least one of the modified sugars comprises 2'-M0E.
50. The antisense oligonucleotide of claim 47, wherein at least one of the modified sugars comprises LNA.
51. The antisense oligonucleotide of claim 46, wherein the backbone linkages of the 5' wing segment, the 3' wing segment, and the gap segment comprise a mixture of phosphorothioate and phosphodiester linkages.
52. A single stranded antisense oligonucleotide that suppresses the expression of a PIKFYVE, wherein the antisense oligonucleotide comprises the sequence set forth in any of SEQ ID NOs: 863-1088, 1092-1110, 1114-1155, 1158-1202, 1205, 1207-1280, 1290, 1336, 1368, 1382-1383, 1401, 1421, 1449, 1455, 1462, 1467, 1481, 1499, 1515-1516, 1531-1532, 1534, 1541, 1544, 1546, 1552, 1554, 1563, 1650, 1652-1654, 1660, 1666- 1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782- 1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895- 1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321, wherein the backbone linkage of the antisense oligonucleotide comprises a mixture of phosphorothioate and phosphodiester linkages.
53. The antisense oligonucleotide of claim 52, wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, sixteenth, and seventeenth linkages are phosphodiester linkages.
54. The antisense oligonucleotide of claim 46, wherein at least one nucleoside comprises a modified nucleobase.
55. The antisense oligonucleotide of claim 46, wherein the modified nucleobase is a 5- methylcytosine.
56. The antisense oligonucleotide of claim 46, wherein the antisense oligonucleotide comprises a moiety that neutralizes charge on the antisense oligonucleotide.
57. The antisense oligonucleotide of claim 46, wherein the antisense oligonucleotide suppresses the expression of a PIKFYVE by at least 80%.
58. A pharmaceutical composition comprising the antisense oligonucleotide of claim 46, and a pharmaceutically acceptable carrier, diluent and/or excipient.
59. A pharmaceutical composition comprising the antisense oligonucleotide of claim 52, and a pharmaceutically acceptable carrier, diluent and/or excipient.
60. A pharmaceutical composition comprising the antisense oligonucleotide of claim 53, and a pharmaceutically acceptable carrier, diluent and/or excipient.
61. The pharmaceutical composition of claim 60, wherein the pharmaceutical composition is formulated for parenteral delivery or intracerebroventricular injection.
62. The pharmaceutical composition of claim 61, wherein the pharmaceutical composition is formulated for parenteral delivery or intracerebroventricular injection.
63. The antisense oligonucleotide of claim 14, wherein the antisense oligonucleotide comprises: a gap segment consisting of 8 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and a 3' wing segment consisting of 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein a nucleoside of each wing segment comprises a modified sugar moiety.
64. The antisense oligonucleotide of claim 14, wherein the antisense oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5' wing segment consisting of 5 linked nucleosides; and a 3' wing segment consisting of 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein a nucleoside of each wing segment comprises a modified sugar moiety.
65. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a nucleobase sequence set forth in any one of SEQ ID NOs: 1-49, wherein the antisense oligonucleotide skips exon 3 of the PIKFYVE protein.
66. The antisense oligonucleotide of claim 65, wherein wherein the oligonucleotide comprise one or more modified sugars.
67. The antisense oligonucleotide of claim 66, wherein the modified sugars comprise 2'-0Me, 2'-M0E, LNA, or any combination thereof.
68. The antisense oligonucleotide of claim 67, wherein at least one of the modified sugars comprises 2'-0Me.
69. The antisense oligonucleotide of claim 67, wherein each nucleoside of the antisense oligonucleotide comprises a 2'-M0E modified sugar.
70. The antisense oligonucleotide of claim 67, wherein at least one of the modified sugars comprises 2'-M0E.
71. The antisense oligonucleotide of claim 67, wherein at least one of the modified sugars comprises LNA.
72. The antisense oligonucleotide of claim 65, wherein the backbone linkages oligonucleotide comprise a mixture of phosphorothioate and phosphodiester linkages.
73. The antisense oligonucleotide of claim 72, wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, sixteenth, and seventeenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, fourteenth, and fifteenth linkages are phosphodiester linkages.
74. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a nucleobase sequence set forth in any one of SEQ ID NOs: 50-80, wherein the antisense oligonucleotide skips exon 5 of the PIKFYVE protein.
75. The antisense oligonucleotide of claim 74, wherein wherein the oligonucleotide comprise one or more modified sugars.
76. The antisense oligonucleotide of claim 75, wherein the modified sugars comprise 2'-0Me, 2'-M0E, LNA, or any combination thereof.
77. The antisense oligonucleotide of claim 76, wherein at least one of the modified sugars comprises 2'-0Me.
78. The antisense oligonucleotide of claim 76, wherein each nucleoside of the antisense oligonucleotide comprises a 2'-M0E modified sugar.
79. The antisense oligonucleotide of claim 76, wherein at least one of the modified sugars comprises 2'-M0E.
80. The antisense oligonucleotide of claim 76, wherein at least one of the modified sugars comprises LNA.
81. The antisense oligonucleotide of claim 74, wherein the backbone linkages oligonucleotide comprise a mixture of phosphorothioate and phosphodiester linkages.
82. The antisense oligonucleotide of claim 81, wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, sixteenth, and seventeenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, fourteenth, and fifteenth linkages are phosphodiester linkages.
83. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a nucleobase sequence set forth in any one of SEQ ID NOs: 81-306, 310-328, 332-373, 376-420, 423, and 425-479, wherein the antisense oligonucleotide comprises a low acute neurotoxicity scores.
84. The antisense oligonucleotide of claim 83, wherein wherein the oligonucleotide comprise one or more modified sugars.
85. The antisense oligonucleotide of claim 84, wherein the modified sugars comprise 2'-0Me, 2'-M0E, LNA, or any combination thereof.
86. The antisense oligonucleotide of claim 85, wherein at least one of the modified sugars comprises 2'-0Me.
87. The antisense oligonucleotide of claim 85, wherein at least one of the modified sugars comprises 2'-M0E.
88. The antisense oligonucleotide of claim 85, wherein at least one of the modified sugars comprises LNA.
89. The antisense oligonucleotide of claim 83, wherein the backbone linkages oligonucleotide comprise a mixture of phosphorothioate and phosphodiester linkages.
90. The antisense oligonucleotide of claim 89, wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, sixteenth, and seventeenth linkages are phosphodiester linkages.
91. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises a nucleobase sequence set forth in any one of SEQ ID NOs: 480-498, 508, 554, 586, 600-601, 619, 639, 667, 673, 680, 685, 699, 717, 733-734, 749-750, 752, 759, 762, 764, 770, 772, 781, and the unmodified versions of SEQ ID NOs: 1650, 1652-1654, 1660, 1666-1667, 1671-1672, 1674, 1677, 1679, 1684-1687, 1689, 1692, 1694, 1696-1698, 1700, 1702-1703, 1708-1711, 1713, 1716-1717, 1719, 1722, 1725, 1727-1728, 1731, 1740, 1743-1745, 1747-1751, 1754, 1756, 1760, 1762-1764, 1770-1775, 1777-1779, 1782-1784, 1787-1788, 1791-1793, 1795, 1797-1802, 1804-1805, 1809-1811, 1813-1815, 1818-1819, 1822-1824, 1826-1827, 1830-1832, 1836-1837, 1840-1843, 1845-1853, 1855-1858, 1861-1862, 1864, 1866, 1868-1869, 1871-1881, 1883, 1885-1892, 1895- 1897, 1899, 1901-1907, 1909, 1911-1913, 1915, 1917, 1919-1927, 1930, 1932-1936, 1938-1940, 1943, 1945-1953, 1955-1969, 1971-1973, 1976-1980, 1985-1994, 1996-1997, 1999-2010, 2012-2015, 2017, 2019-2021, 2023, 2026-2029, 2031-2042, 2044-2055, 2058, 2061-2070, 2072-2083, 2085-2090, 2092-2095, 2097, 2099-2104, 2106-2109, 2111-2116, 2118, 2120-2121, 2123-2124, 2126-2128, 2130, 2132-2143, 2146-2147, 2149-2164, 2166-2177, 2179-2183, 2185, 2187-2205, 2207-2216, 2218, 2220-2232, 2234, 2236-2245, 2247-2268, 2270-2289, 2291-2300, 2302, 2304-2316, and 2319-2321, wherein the antisense oligonucleotide comprises a low numbers of off-target binding sites.
92. The antisense oligonucleotide of claim 91, wherein wherein the oligonucleotide comprise one or more modified sugars.
93. The antisense oligonucleotide of claim 92, wherein the modified sugars comprise 2'-0Me, 2'-M0E, LNA, or any combination thereof.
94. The antisense oligonucleotide of claim 93, wherein at least one of the modified sugars comprises 2'-0Me.
95. The antisense oligonucleotide of claim 93, wherein at least one of the modified sugars comprises 2'-M0E.
96. The antisense oligonucleotide of claim 93, wherein at least one of the modified sugars comprises LNA.
97. The antisense oligonucleotide of claim 91, wherein the backbone linkages oligonucleotide comprise a mixture of phosphorothioate and phosphodiester linkages.
98. The antisense oligonucleotide of claim 97, wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth linkages are phosphorothioate linkages, and the second, third, fourth, fifth, sixteenth, and seventeenth linkages are phosphodiester linkages.
EP24713778.9A 2023-02-06 2024-02-06 Pikfyve antisense oligonucleotides Pending EP4662318A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363483517P 2023-02-06 2023-02-06
PCT/US2024/014647 WO2024167945A1 (en) 2023-02-06 2024-02-06 Pikfyve antisense oligonucleotides

Publications (1)

Publication Number Publication Date
EP4662318A1 true EP4662318A1 (en) 2025-12-17

Family

ID=90458656

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24713778.9A Pending EP4662318A1 (en) 2023-02-06 2024-02-06 Pikfyve antisense oligonucleotides

Country Status (8)

Country Link
EP (1) EP4662318A1 (en)
JP (1) JP2026504486A (en)
KR (1) KR20250143312A (en)
CN (1) CN120659879A (en)
AU (1) AU2024217638A1 (en)
IL (1) IL322380A (en)
MX (1) MX2025009152A (en)
WO (1) WO2024167945A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120350010B (en) * 2025-06-24 2025-09-09 羿美诚健(上海)生物医药有限公司 Small nucleic acid targeting PIKfyve gene and pharmaceutical composition and use thereof

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
USRE44779E1 (en) 1997-03-07 2014-02-25 Santaris Pharma A/S Bicyclonucleoside and oligonucleotide analogues
JP3756313B2 (en) 1997-03-07 2006-03-15 武 今西 Novel bicyclonucleosides and oligonucleotide analogues
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
JP4236812B2 (en) 1997-09-12 2009-03-11 エクシコン エ／エス Oligonucleotide analogues
US7572582B2 (en) 1997-09-12 2009-08-11 Exiqon A/S Oligonucleotide analogues
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US20030104410A1 (en) * 2001-03-16 2003-06-05 Affymetrix, Inc. Human microarray
AU2003291755A1 (en) 2002-11-05 2004-06-07 Isis Pharmaceuticals, Inc. Oligomers comprising modified bases for binding cytosine and uracil or thymine and their use
US7250289B2 (en) * 2002-11-20 2007-07-31 Affymetrix, Inc. Methods of genetic analysis of mouse
WO2004106356A1 (en) 2003-05-27 2004-12-09 Syddansk Universitet Functionalized nucleotide derivatives
AU2007249349B2 (en) 2006-05-11 2012-03-08 Isis Pharmaceuticals, Inc. 5'-Modified bicyclic nucleic acid analogs
US7666854B2 (en) 2006-05-11 2010-02-23 Isis Pharmaceuticals, Inc. Bis-modified bicyclic nucleic acid analogs
WO2009067647A1 (en) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
WO2009100320A2 (en) 2008-02-07 2009-08-13 Isis Pharmaceuticals, Inc. Bicyclic cyclohexitol nucleic acid analogs
DK2356129T3 (en) 2008-09-24 2013-05-13 Isis Pharmaceuticals Inc Substituted alpha-L bicyclic nucleosides
US9012421B2 (en) 2009-08-06 2015-04-21 Isis Pharmaceuticals, Inc. Bicyclic cyclohexose nucleic acid analogs
US10758545B2 (en) 2015-06-25 2020-09-01 University Of Southern California Methods to treat neurological diseases
IL320434A (en) 2015-07-22 2025-06-01 Wave Life Sciences Ltd Oligonucleotide compositions and methods thereof
WO2021155067A1 (en) * 2020-01-28 2021-08-05 University Of Southern California Compositions and methods to treat neurological diseases
EP3960875A1 (en) * 2020-08-28 2022-03-02 Koninklijke Philips N.V. Pcr method and kit for determining pathway activity
US12104159B2 (en) * 2021-06-22 2024-10-01 AcuraStem Incorporated PIKFYVE antisense oligonucleotides

Also Published As

Publication number Publication date
WO2024167945A1 (en) 2024-08-15
AU2024217638A1 (en) 2025-07-31
IL322380A (en) 2025-09-01
JP2026504486A (en) 2026-02-05
MX2025009152A (en) 2025-09-02
KR20250143312A (en) 2025-10-01
CN120659879A (en) 2025-09-16

Similar Documents

Publication Publication Date Title
US12104155B2 (en) UNC13A antisense oligonucleotides
US12104159B2 (en) PIKFYVE antisense oligonucleotides
US20230098111A1 (en) Compositions and methods to treat neurological diseases
WO2024155986A2 (en) Unc13a antisense oligonucleotides
US20230066380A1 (en) Antagonism as a therapy for tdp-43 proteinopathies
AU2024217638A1 (en) Pikfyve antisense oligonucleotides
US20250223591A1 (en) Syf2 antisense oligonucleotides
KR20250138204A (en) SYF2 antisense oligonucleotide
EA050711B1 (en) Antisense Oligonucleotides Targeting PIKFYVE
CN118475695A (en) UNC13A antisense oligonucleotides
KR20250163337A (en) Treatment of nervous system disorders

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250828

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR